Biosynthetic Polypeptide Fusion Inhibitors

ABSTRACT

Modified biosynthetic polypeptide fusion inhibitors, methods for manufacturing, and uses thereof are provided.

FIELD OF THE INVENTION

This invention relates to biosynthetic polypeptides and fusion proteinsthat inhibit membrane fusion events, and comprise or are made utilizingat least one non-naturally-encoded amino acid.

BACKGROUND OF THE INVENTION

Respiratory Syncytial Virus (RSV) belongs to Paramyxoviridae family. RSVis the major cause of lower respiratory infections in infants, elderly,and immuno-compromised individuals, including but not limited to,transplantation patients. There is still no effective treatment or avaccine. RSV is a single stranded negative sense RNA virus that encodesfor 11 proteins, 9 of them are structural proteins and 2 of them areregulatory proteins for viral replication. RSV contains two majorsurface glycoproteins, the receptor-binding protein (G), which allowsthe virus to attach to the host receptor, and the fusion (F) protein,which enables the virus to enter the host cell. Fusion of the RSVenvelope, which occurs at neutral pH, induces a vast syncytia formationbetween the infected cells with the bystander uninfected cells. The Fprotein is cleaved to generate two disulfide-linked polypeptides namedF1 from the C terminus and the F2 from the N terminus. Adjacent to thesetwo regions are two heptad repeat sequences named HR-C and HR-N thatform a trimer of hairpin-like structures which allows fusion between theviral and the host cell membranes. The heptad region is a potentialtarget for designing inhibitor peptides that bind to HR-N and thereforeprevent the hairpin-structure formation and subsequent fusion. The RSVfusion process is very similar to the HIV fusion mechanism.

Two independent lines of research have focused on the development ofpeptides that inhibit RSV entry. Peptides derived from the HR-C regionare similar in concept to the product FUZEON® (Roche) which blocks HIVfusion. Peptides with a general anionic character, such as those derivedfrom GTPase RhoA, have also demonstrated anti-viral activity againstRSV. These RhoA derived peptides apparently act through a separatemechanism of inhibiting viral cell surface contact.

Peptides are widely used in research and medical practice, and it can beexpected that their importance will increase as challenges tomanufacturing and performance of the peptide products are addressed.Therapeutic peptides such as those described herein are referred to asbiosynthetic polypeptide fusion inhibitors (BPFIs).

When native peptides or analogues thereof are used in therapy, it isgenerally found that they have a high rate of degradation and/orclearance. A high rate of clearance of a therapeutic agent isinconvenient in cases where it is desired to maintain a high blood levelthereof over a prolonged period of time since repeated administrationswill then be necessary. In some cases it is possible to influence therelease profile of peptides by applying suitable pharmaceuticalcompositions, but this approach has various shortcomings and is notgenerally applicable.

Peptidases break a peptide bond in peptides by inserting a watermolecule across the bond. Generally, most peptides are broken down bypeptidases in the body in a manner of a few minutes or less. Inaddition, some peptidases are specific for certain types of peptides,making their degradation even more rapid. Thus, if a peptide is used asa therapeutic agent, its activity is generally reduced as the peptidequickly degrades in the body due to the action of peptidases.

One way to overcome this disadvantage is to administer large dosages ofthe therapeutic peptide of interest to the patient so that even if someof the peptide is degraded, enough remains to be therapeuticallyeffective. However, this method is quite uncomfortable for the patient.Since most therapeutic peptides cannot be administered orally, thetherapeutic peptide would have to be either constantly infused,frequently administered by intravenous injections, or administeredfrequently by the inconvenient route of subcutaneous injections. Theneed for frequent administration also results in an unacceptably highprojected cost per treatment course for many potential peptidetherapeutics. The presence of large amounts of degraded peptide may alsogenerate undesired side effects.

Covalent attachment of the hydrophilic polymer poly(ethylene glycol),abbreviated PEG, is a method of increasing water solubility,bioavailability, increasing serum half-life, increasing therapeutichalf-life, modulating immunogenicity, modulating biological activity, orextending the circulation time of many biologically active molecules,including proteins, peptides, and particularly hydrophobic molecules.PEG has been used extensively in pharmaceuticals, on artificialimplants, and in other applications where biocompatibility, lack oftoxicity, and lack of immunogenicity are of importance. In order tomaximize the desired properties of PEG, the total molecular weight andhydration state of the PEG polymer or polymers attached to thebiologically active molecule must be sufficiently high to impart theadvantageous characteristics typically associated with PEG polymerattachment, such as increased water solubility and circulating halflife, while not adversely impacting the bioactivity of the parentmolecule.

PEG derivatives are frequently linked to biologically active moleculesthrough reactive chemical functionalities, such as lysine, cysteine andhistidine residues, the N-terminus and carbohydrate moieties. Proteinsand other molecules often have a limited number of reactive sitesavailable for polymer attachment. Often, the sites most suitable formodification via polymer attachment play a significant role in receptorbinding, and are necessary for retention of the biological activity ofthe molecule. As a result, indiscriminate attachment of polymer chainsto such reactive sites on a biologically active molecule often leads toa significant reduction or even total loss of biological activity of thepolymer-modified molecule. R. Clark et al., (1996), J. Biol. Chem.,271:21969-21977. To form conjugates having sufficient polymer molecularweight for imparting the desired advantages to a target molecule, priorart approaches have typically involved random attachment of numerouspolymer arms to the molecule, thereby increasing the risk of a reductionor even total loss in bioactivity of the parent molecule.

Reactive sites that form the loci for attachment of PEG derivatives toproteins are dictated by the protein's structure. Proteins, includingenzymes, are composed of various sequences of alpha-amino acids, whichhave the general structure H₂N—CHR—COOH. The alpha amino moiety (H₂N—)of one amino acid joins to the carboxyl moiety (—COOH) of an adjacentamino acid to form amide linkages, which can be represented as—(NH—CHR—CO)_(n)—, where the subscript “n” can equal hundreds orthousands. The fragment represented by R can contain reactive sites forprotein biological activity and for attachment of PEG derivatives.

For example, in the case of the amino acid lysine, there exists an —NH₂moiety in the epsilon position as well as in the alpha position. Theepsilon —NH₂ is free for reaction under conditions of basic pH. Much ofthe art in the field of protein derivatization with PEG has beendirected to developing PEG derivatives for attachment to the epsilon—NH₂ moiety of lysine residues present in proteins. “Polyethylene Glycoland Derivatives for Advanced PEGylation”, Nektar Molecular EngineeringCatalog, 2003, pp. 1-17. These PEG derivatives all have the commonlimitation, however, that they cannot be installed selectively among theoften numerous lysine residues present on the surfaces of proteins. Thiscan be a significant limitation in instances where a lysine residue isimportant to protein activity, existing in an enzyme active site forexample, or in cases where a lysine residue plays a role in mediatingthe interaction of the protein with other biological molecules, as inthe case of receptor binding sites.

A second and equally important complication of existing methods forprotein PEGylation is that the PEG derivatives can undergo undesiredside reactions with residues other than those desired. Histidinecontains a reactive imino moiety, represented structurally as —N(H)—,but many chemically reactive species that react with epsilon —NH₂ canalso react with —N(H)—. Similarly, the side chain of the amino acidcysteine bears a free sulfhydryl group, represented structurally as —SH.In some instances, the PEG derivatives directed at the epsilon —NH₂group of lysine also react with cysteine, histidine or other residues.This can create complex, heterogeneous mixtures of PEG-derivatizedbioactive molecules and risks destroying the activity of the bioactivemolecule being targeted. It would be desirable to develop PEGderivatives that permit a chemical functional group to be introduced ata single site within the protein that would then enable the selectivecoupling of one or more PEG polymers to the bioactive molecule atspecific sites on the protein surface that are both well-defined andpredictable.

In addition to lysine residues, considerable effort in the art has beendirected toward the development of activated PEG reagents that targetother amino acid side chains, including cysteine, histidine and theN-terminus. See, e.g., U.S. Pat. No. 6,610,281 which is incorporated byreference herein, and “Polyethylene Glycol and Derivatives for AdvancedPEGylation”, Nektar Molecular Engineering Catalog, 2003, pp. 1-17. Acysteine residue can be introduced site-selectively into the structureof proteins using site-directed mutagenesis and other techniques knownin the art, and the resulting free sulfhydryl moiety can be reacted withPEG derivatives that bear thiol-reactive functional groups. Thisapproach is complicated, however, in that the introduction of a freesulfhydryl group can complicate the expression, folding and stability ofthe resulting protein. Thus, it would be desirable to have a means tointroduce a chemical functional group into bioactive molecules thatenables the selective coupling of one or more PEG polymers to theprotein while simultaneously being compatible with (i.e., not engagingin undesired side reactions with) sulfhydryls and other chemicalfunctional groups typically found in proteins.

As can be seen from a sampling of the art, many of these derivativesthat have been developed for attachment to the side chains of proteins,in particular, the —NH₂ moiety on the lysine amino acid side chain andthe —SH moiety on the cysteine side chain, have proven problematic intheir synthesis and use. Some form unstable linkages with the proteinthat are subject to hydrolysis and therefore decompose, degrade, or areotherwise unstable in aqueous environments, such as in the bloodstream.Some form more stable linkages, but are subject to hydrolysis before thelinkage is formed, which means that the reactive group on the PEGderivative may be inactivated before the protein can be attached. Someare somewhat toxic and are therefore less suitable for use in vivo. Someare too slow to react to be practically useful. Some result in a loss ofprotein activity by attaching to sites responsible for the protein'sactivity. Some are not specific in the sites to which they will attach,which can also result in a loss of desirable activity and in a lack ofreproducibility of results. In order to overcome the challengesassociated with modifying proteins with poly(ethylene glycol) moieties,PEG derivatives have been developed that are more stable (e.g., U.S.Pat. No. 6,602,498, which is incorporated by reference herein) or thatreact selectively with thiol moieties on molecules and surfaces (e.g.,U.S. Pat. No. 6,610,281, which is incorporated by reference herein).There is clearly a need in the art for PEG derivatives that arechemically inert in physiological environments until called upon toreact selectively to form stable chemical bonds.

Recently, an entirely new technology in the protein sciences has beenreported, which promises to overcome many of the limitations associatedwith site-specific modifications of proteins. Specifically, newcomponents have been added to the protein biosynthetic machinery of theprokaryote Escherichia coli (E. coli) (e.g., L. Wang, et al., (2001),Science 292:498-500) and the eukaryote Sacchromyces cerevisiae (S.cerevisiae) (e.g., J. Chin et al., Science 301:964-7 (2003)), which hasenabled the incorporation of non-genetically encoded amino acids toproteins in vivo. A number of new amino acids with novel chemical,physical or biological properties, including photoaffinity labels andphotoisomerizable amino acids, keto amino acids, and glycosylated aminoacids have been incorporated efficiently and with high fidelity intoproteins in E. coli and in yeast in response to the amber codon, TAG,using this methodology. See, e.g., J. W. Chin et al., (2002), Journal ofthe American Chemical Society 124:9026-9027; J. W. Chin, & P. G.Schultz, (2002), ChemBioChem 3(11):1135-1137; J. W. Chin, et al.,(2002), PNAS United States of America 99:11020-11024; and, L. Wang, & P.G. Schultz, (2002), Chem. Comm., 1:1-11. These studies have demonstratedthat it is possible to selectively and routinely introduce chemicalfunctional groups, such as ketone groups, alkyne groups and azidemoieties, that are not found in proteins, that are chemically inert toall of the functional groups found in the 20 common, genetically-encodedamino acids and that may be used to react efficiently and selectively toform stable covalent linkages.

The ability to incorporate non-genetically encoded amino acids intoproteins permits the introduction of chemical functional groups thatcould provide valuable alternatives to the naturally-occurringfunctional groups, such as the epsilon —NH₂ of lysine, the sulfhydryl—SH of cysteine, the imino group of histidine, etc. Certain chemicalfunctional groups are known to be inert to the functional groups foundin the 20 common, genetically-encoded amino acids but react cleanly andefficiently to form stable linkages. Azide and acetylene groups, forexample, are known in the art to undergo a Huisgen [3+2] cycloadditionreaction in aqueous conditions in the presence of a catalytic amount ofcopper. See, e.g., Tornoe, et al., (2002) J. Org. Chem. 67:3057-3064;and, Rostovtsev, et al., (2002) Angew. Chem. Int. Ed. 41:2596-2599. Byintroducing an azide moiety into a protein structure, for example, oneis able to incorporate a functional group that is chemically inert toamines, sulfhydryls, carboxylic acids, hydroxyl groups found inproteins, but that also reacts smoothly and efficiently with anacetylene moiety to form a cycloaddition product. Importantly, in theabsence of the acetylene moiety, the azide remains chemically inert andunreactive in the presence of other protein side chains and underphysiological conditions.

The present invention addresses, among other things, problems associatedwith the activity and production of BPFI's, and also addresses theproduction of a BPFI with improved biological or pharmacologicalproperties, such as improved therapeutic half-life.

BRIEF SUMMARY OF THE INVENTION

The present invention provides RSV entry inhibitors having an improvedhelical propensity of HR-C derived peptides. The present invention alsoprovides RSV entry inhibitors having a combination of the activity offusion inhibitors and anionic peptide activities. The present inventionalso provides BPFI's having site-specific PEGylation to improve thepharmacological properties of the peptides. This invention providesbiosynthetic peptide fusion inhibitors (BPFIs) including, but notlimited to, membrane fusion inhibitory peptides and anionic peptides,comprising one or more non-naturally encoded amino acids. Any BPFI,fragment, analog, or variant thereof with therapeutic activity may beused in this invention. Numerous examples of BPFIs that may be used inthis invention have been provided. However, the lists provided are notexhaustive and in no way limit the number or type of BPFIs that may beused in this invention. Thus, any BPFI and/or fragments, analogs, andvariants produced from any BPFI including novel BPFIs may be modifiedaccording to the present invention, and used therapeutically.

In some embodiments, the BPFI comprises one or more post-translationalmodifications. In some embodiments, the BPFI is linked to a linker,polymer, or biologically active molecule. In some embodiments, the BPFIis linked to a bifunctional polymer, bifunctional linker, or at leastone additional BPFI.

In some embodiments, the non-naturally encoded amino acid is linked to awater soluble polymer. In some embodiments, the water soluble polymercomprises a poly(ethylene glycol) moiety. In some embodiments, thepoly(ethylene glycol) molecule is a bifunctional polymer. In someembodiments, the bifunctional polymer is linked to a second polypeptide.In some embodiments, the second polypeptide is a BPFI.

In some embodiments, the non-naturally encoded amino acid is linked to awater soluble polymer. In some embodiments, the non-naturally encodedamino acid is linked to the water soluble polymer with a linker orbonded to the water soluble polymer. In some embodiments, thenon-naturally encoded amino acid is linked to the water soluble polymerwith a linker that is biodegradable. In some embodiments, thebiodegradable linker can be used to form a prodrug comprising the BPFI.In one example of this prodrug approach, the water soluble polymerblocks BPFI activity, and degradation of the linker releases activeBPFI. In some embodiments, the non-naturally encoded amino acid islinked to an acyl moiety or acyl chain. In some embodiments, thenon-naturally encoded amino acid is linked to an acyl moiety or acylchain by a linker. In some embodiments, the non-naturally encoded aminoacid is linked to an acyl moiety or acyl chain by a poly(ethyleneglycol) linker or a prodrug. In some embodiments, the non-naturallyencoded amino acid is linked to serum albumin. In some embodiments, thenon-naturally encoded amino acid is linked to serum albumin by a linker.In some embodiments, the linker is a poly(ethylene glycol) or a prodrug.In some embodiments, the linker is a dual cleavage prodrug in which step1 is controlled release of a molecule such as albumin and step 2 is asecond cleavage releasing the linker or a portion thereof.

In some embodiments, the BPFI comprises an intramolecular bridge betweentwo amino acids present in the BPFI. In some embodiments, the BPFIcomprises one or more non-naturally encoded amino acids. One of the twobridged residues may be a non-naturally encoded amino acid or anaturally encoded amino acid. The non-natural amino acids may be joinedby a linker, polymer, or a biologically active molecule.

In some embodiments, the BPFI comprises at least two amino acids linkedto a water soluble polymer comprising a poly(ethylene glycol) moiety. Insome embodiments, at least one amino acid is a non-naturally encodedamino acid.

In some embodiments, one or more non-naturally encoded amino acids areincorporated at any position in the BPFI, such as HR-C, HR-N or anionicpeptide, a fusion of any one or more of these peptides, or a fragment ofany one or more of these peptides, before the first amino acid (at theamino terminus), an addition at the carboxy terminus, or any combinationthereof. In some embodiments, one or more non-naturally encoded aminoacids are incorporated at any position within the amino acid sequence ofthe BPFI.

In some embodiments, the non-naturally occurring amino acid at one ormore of these positions is linked to a water soluble polymer.

In some embodiments, the BPFI polypeptides of the invention comprise oneor more non-naturally occurring amino acids at one or more amino acidpositions adjacent to or within the BPFI sequence providing anantagonist.

In some embodiments, the BPFI comprises a substitution, addition ordeletion that modulates affinity of the BPFI for a BPFI receptor or abinding partner, including, but not limited to, a protein, polypeptide,small molecule, lipid, or nucleic acid. In some embodiments, the BPFIcomprises a substitution, addition, or deletion that increases thestability of the BPFI when compared with the stability of thecorresponding BPFI without the substitution, addition, or deletion. Insome embodiments, the BPFI comprises a substitution, addition, ordeletion that modulates the immunogenicity of the BPFI when comparedwith the immunogenicity of the corresponding BPFI without thesubstitution, addition, or deletion. In some embodiments, the BPFIcomprises a substitution, addition, or deletion that modulates serumhalf-life or circulation time of the BPFI when compared with the serumhalf-life or circulation time of the corresponding BPFI without thesubstitution, addition, or deletion.

In some embodiments, the BPFI comprises a substitution, addition, ordeletion that increases the aqueous solubility of BPFI when comparedwith the aqueous solubility of the corresponding BPFI without thesubstitution, addition, or deletion. In some embodiments, the BPFIcomprises a substitution, addition, or deletion that increases thesolubility of the BPFI produced in a host cell when compared with thesolubility of the corresponding BPFI without the substitution, addition,or deletion. In some embodiments, the BPFI comprises a substitution,addition, or deletion that increases the expression of the BPFI in ahost cell or increases synthesis in vitro when compared with theexpression or synthesis of the corresponding BPFI without thesubstitution, addition, or deletion. In some embodiments, the BPFIcomprises a substitution, addition, or deletion that decreases peptidaseor protease susceptibility of the BPFI when compared with the peptidaseor protease susceptibility of the corresponding BPFI without thesubstitution, addition, or deletion. In some embodiments, the BPFIcomprises a substitution, addition, or deletion that modulates signaltransduction activity of the BPFI receptor or binding partner whencompared with the activity of the corresponding BPFI without thesubstitution, addition, or deletion. In some embodiments, the BPFIcomprises a substitution, addition, or deletion that modulates itsbinding to another molecule such as a receptor when compared with thebinding of the corresponding BPFI without the substitution, addition, ordeletion. In some embodiments, the BPFI comprises a substitution,addition, or deletion that modulates the conformation or one or morebiological activities of its binding partner when compared with thebinding partner's conformation or biological activity after binding ofcorresponding BPFI without the substitution, addition, or deletion.

In some embodiments the amino acid substitutions in the BPFI may be withnaturally occurring or non-naturally occurring amino acids, providedthat at least one substitution is with a non-naturally encoded aminoacid.

In some embodiments, the non-naturally encoded amino acid comprises acarbonyl group, an aminooxy group, a hydrazine group, a hydrazide group,a semicarbazide group, an azide group, or an alkyne group.

In some embodiments, the non-naturally encoded amino acid comprises acarbonyl group. In some embodiments, the non-naturally encoded aminoacid has the structure:

wherein n is 0-10; R₁ is an alkyl, aryl, substituted alkyl, orsubstituted aryl; R₂ is H, an alkyl, aryl, substituted alkyl, andsubstituted aryl; and R₃ is H, an amino acid, a polypeptide, or an aminoterminus modification group, and R₄ is H, an amino acid, a polypeptide,or a carboxy terminus modification group.

In some embodiments, the non-naturally encoded amino acid comprises anaminooxy group. In some embodiments, the non-naturally encoded aminoacid comprises a hydrazide group. In some embodiments, the non-naturallyencoded amino acid comprises a hydrazine group. In some embodiments, thenon-naturally encoded amino acid residue comprises a semicarbazidegroup.

In some embodiments, the non-naturally encoded amino acid residuecomprises an azide group. In some embodiments, the non-naturally encodedamino acid has the structure:

wherein n is 0-10; R₁ is an alkyl, aryl, substituted alkyl, substitutedaryl or not present; X is O, N, S or not present; m is 0-10; R₂ is H, anamino acid, a polypeptide, or an amino terminus modification group, andR₃ is H, an amino acid, a polypeptide, or a carboxy terminusmodification group.

In some embodiments, the non-naturally encoded amino acid comprises analkyne group. In some embodiments, the non-naturally encoded amino acidhas the structure:

wherein n is 0-10; R₁ is an alkyl, aryl, substituted alkyl, orsubstituted aryl; X is O, N, S or not present; m is 0-10, R₂ is H, anamino acid, a polypeptide, or an amino terminus modification group, andR₃ is H, an amino acid, a polypeptide, or a carboxy terminusmodification group.

In some embodiments, the polypeptide is a BPFI agonist, partial agonist,antagonist, partial antagonist, or inverse agonist. In some embodiments,the BPFI agonist, partial agonist, antagonist, partial antagonist, orinverse agonist comprises a non-naturally encoded amino acid linked to awater soluble polymer. In some embodiments, the water soluble polymercomprises a poly(ethylene glycol) moiety. In some embodiments, the BPFIagonist, partial agonist, antagonist, partial antagonist, or inverseagonist comprises a non-naturally encoded amino acid and one or morepost-translational modification, linker, polymer, or biologically activemolecule. In some embodiments, the non-naturally encoded amino acidlinked to a water soluble polymer is present within the receptor bindingregion of the BPFI or interferes with the receptor binding of the BPFI.In some embodiments, the non-naturally encoded amino acid linked to awater soluble polymer is present within the region of the BPFI thatbinds to a binding partner or interferes with the binding of a bindingpartner to the BPFI.

The present invention also provides isolated nucleic acids comprising apolynucleotide that hybridizes under stringent conditions to anucleotide sequence encoding a polypeptide having the amino acidsequence in SEQ ID NO: 1 wherein the polynucleotide comprises at leastone selector codon. In some embodiments, the selector codon is selectedfrom the group consisting of an amber codon, ochre codon, opal codon, aunique codon, a rare codon, and a four-base codon.

The present invention also provides methods of making a BPFI linked to awater soluble polymer. In some embodiments, the method comprisescontacting an isolated BPFI comprising a non-naturally encoded aminoacid with a water soluble polymer comprising a moiety that reacts withthe non-naturally encoded amino acid. In some embodiments, thenon-naturally encoded amino acid incorporated into the BPFI is reactivetoward a water soluble polymer that is otherwise unreactive toward anyof the 20 common amino acids. In some embodiments, the non-naturallyencoded amino acid incorporated into the BPFI is reactive toward alinker, polymer, or biologically active molecule that is otherwiseunreactive toward any of the 20 common amino acids.

In some embodiments, the BPFI linked to the water soluble polymer ismade by reacting a BPFI comprising a carbonyl-containing amino acid witha poly(ethylene glycol) molecule comprising an aminooxy, hydrazine,hydrazide or semicarbazide group. In some embodiments, the aminooxy,hydrazine, hydrazide or semicarbazide group is linked to thepoly(ethylene glycol) molecule through an amide linkage.

In some embodiments, the BPFI linked to the water soluble polymer ismade by reacting a poly(ethylene glycol) molecule comprising a carbonylgroup with a BPFI comprising a non-naturally encoded amino acid thatcomprises an aminooxy, hydrazine, hydrazide or semicarbazide group.

In some embodiments, the BPFI linked to the water soluble polymer ismade by reacting a BPFI comprising an alkyne-containing amino acid witha poly(ethylene glycol) molecule comprising an azide moiety. In someembodiments, the azide or alkyne group is linked to the poly(ethyleneglycol) molecule through an amide linkage.

In some embodiments, the BPFI linked to the water soluble polymer ismade by reacting a BPFI comprising an azide-containing amino acid with apoly(ethylene glycol) molecule comprising an alkyne moiety. In someembodiments, the azide or alkyne group is linked to the poly(ethyleneglycol) molecule through an amide linkage.

In some embodiments, the poly(ethylene glycol) molecule has a molecularweight of between about 0.1 kDa and about 100 kDa. In some embodiments,the poly(ethylene glycol) molecule has a molecular weight of between 0.1kDa and 50 kDa.

In some embodiments, the poly(ethylene glycol) molecule is a branchedpolymer. In some embodiments, each branch of the poly(ethylene glycol)branched polymer has a molecular weight of between 1 kDa and 100 kDa, orbetween 1 kDa and 50 kDa.

In some embodiments, the water soluble polymer linked to BPFI comprisesa polyalkylene glycol moiety. In some embodiments, the non-naturallyencoded amino acid residue incorporated into BPFI comprises a carbonylgroup, an aminooxy group, a hydrazide group, a hydrazine, asemicarbazide group, an azide group, or an alkyne group. In someembodiments, the non-naturally encoded amino acid residue incorporatedinto BPFI comprises a carbonyl moiety and the water soluble polymercomprises an aminooxy, hydrazide, hydrazine, or semicarbazide moiety. Insome embodiments, the non-naturally encoded amino acid residueincorporated into BPFI comprises an alkyne moiety and the water solublepolymer comprises an azide moiety. In some embodiments, thenon-naturally encoded amino acid residue incorporated into BPFIcomprises an azide moiety and the water soluble polymer comprises analkyne moiety.

The present invention also provides compositions comprising a BPFIcomprising a non-naturally encoded amino acid and a pharmaceuticallyacceptable carrier. In some embodiments, the non-naturally encoded aminoacid is linked to a water soluble polymer.

The present invention also provides cells comprising a polynucleotideencoding the BPFI comprising a selector codon. In some embodiments, thecells comprise an orthogonal RNA synthetase and/or an orthogonal tRNAfor substituting a non-naturally encoded amino acid into the BPFI.

The present invention also provides methods of making a BPFI comprisinga non-naturally encoded amino acid. In some embodiments, the methodscomprise culturing cells comprising a polynucleotide or polynucleotidesencoding a BPFI, an orthogonal RNA synthetase and/or an orthogonal tRNAunder conditions to permit expression of the BPFI; and purifying theBPFI from the cells and/or culture medium.

The present invention also provides methods of increasing therapeutichalf-life, serum half-life or circulation time of BPFI. The presentinvention also provides methods of modulating immunogenicity of BPFI. Insome embodiments, the methods comprise substituting a non-naturallyencoded amino acid for any one or more amino acids in naturallyoccurring BPFI and/or linking the BPFI to a linker, a polymer, a watersoluble polymer, or a biologically active molecule.

The present invention also provides methods of treating a patient inneed of such treatment with an effective amount of a BPFI of the presentinvention. In some embodiments, the methods comprise administering tothe patient a therapeutically-effective amount of a pharmaceuticalcomposition comprising a BPFI comprising a non-naturally-encoded aminoacid and a pharmaceutically acceptable carrier. In some embodiments, thenon-naturally encoded amino acid is linked to a water soluble polymer.

The present invention provides a BPFI comprising at least one linker,polymer, or biologically active molecule, wherein said linker, polymer,or biologically active molecule is attached to the polypeptide through afunctional group of a non-naturally encoded amino acid ribosomallyincorporated into the polypeptide. In some embodiments, the BPFI ismonoPEGylated. The present invention also provides a BPFI comprising alinker, polymer, or biologically active molecule that is attached to oneor more non-naturally encoded amino acid wherein said non-naturallyencoded amino acid is ribosomally incorporated into the polypeptide atpre-selected sites.

In another embodiment, conjugation of the BPFI comprising one or morenon-naturally occurring amino acids to another molecule, including butnot limited to PEG, provides substantially purified BPFI due to theunique chemical reaction utilized for conjugation to the non-naturalamino acid. Conjugation of BPFI comprising one or more non-naturallyencoded amino acids to another molecule, such as PEG, may be performedwith other purification techniques performed prior to or following theconjugation step to provide substantially pure BPFI.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1—The cloning of T20 and TEX is shown.

FIG. 2—Strategy for producing a BPFI is shown.

FIG. 3—A helical analysis of TEX is shown.

FIG. 4—Suppression of a selector codon to incorporate a non-naturallyencoded aino acid is shown.

FIG. 5—Cleavage of peptide by CNBr to provide BPFI is shown.

FIG. 6—A BPFI activity assay is shown.

FIG. 7 Panel A and 7 Panel B—BPFI inhibition of viral infectivity isshown.

FIG. 8—Conjugation of BPFI with PEG is shown.

FIG. 9—Constructs for incorporation of a non-naturally encoded aminoacid into T-20 and TEX are shown (FIG. 9, Panel A). FIG. 9, Panel Bshows T-20 polypeptides before and after CNBr cleavage.

FIG. 10—A comparison of wild-type T-20 and TEX sequences is shown inFIG. 10, and residues encoded by codons that were substituted with anamber codon are marked with an asterisk.

FIG. 11—An in vitro fusion assay to test T-20 and TEX antiviral activityis shown.

FIG. 12 Panel A and 12 Panel B—Coomassie stained polyacrylamide gels ofT20 651 suppression (FIG. 12, Panel A) and TEX 636 suppression (FIG. 12,Panel B) are shown. Westerns (anti-His) of the samples shown in Panel Aand B are shown in FIG. 12, Panels C and D. FIG. 12, Panel E shows theresidues substituted with p-acetyl-phenylalanine with asterisks in T-20(T-20-Mut651) and in TEX (TEX-Mut636).

FIG. 13—A diagram of the RSV F protein with a peptide fusion inhibitoris shown.

DEFINITIONS

It is to be understood that this invention is not limited to theparticular methodology, protocols, cell lines, constructs, and reagentsdescribed herein and as such may vary. It is also to be understood thatthe terminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention, which will be limited only by the appended claims.

As used herein and in the appended claims, the singular forms “a,” “an,”and “the” include plural reference unless the context clearly indicatesotherwise. Thus, for example, reference to a “BPFI” is a reference toone or more such polypeptides and includes equivalents thereof known tothose skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention belongs. Although any methods, devices,and materials similar or equivalent to those described herein can beused in the practice or testing of the invention, the preferred methods,devices and materials are now described.

All publications and patents mentioned herein are incorporated herein byreference for the purpose of describing and disclosing, for example, theconstructs and methodologies that are described in the publications,which might be used in connection with the presently describedinvention. The publications discussed herein are provided solely fortheir disclosure prior to the filing date of the present application.Nothing herein is to be construed as an admission that the inventors arenot entitled to antedate such disclosure by virtue of prior invention orfor any other reason.

A “BPFI” refers to a polymer of amino acid residues covalently linked bypeptide bonds that is produced from an mRNA with a selector codon. BPFIsinclude, but are not limited to, HR-C, HR-N and anionic peptides. A BPFImay be a fragment of a polymer that is greater than about 100 aminoacids in length and may or may not include additional amino acids suchas, but not limited to, a leader sequence or secretion signal sequence.BPFIs includes peptides comprising a fragment of the HR-C region, afragment of the HR-N region, or fragment of anionic peptides or anycombination thereof. BPFIs also include a heterodimeric or multimericpeptide comprising one or more HR-C derived peptide and anionic peptide.BPFI molecules include fusions. Such fusion include but are not limitedto: RhoA peptide—amino acid linker—HR-C peptide; HR-C peptide—amino acidlinker—RhoA peptide. Spacers may be variable in size, and include, butis not limited to, a Gly-Ser linker. A linker itself may contain anon-naturally encoded amino acid. A non-naturally encoded amino acid maybe substituted in the RhoA peptide or the HR-C peptide for attachment ofmolecules including but not limited to, polymers, biologically activemolecules, PEG or other chemical linkers. A linker may also be T shaped,connecting the RhoA peptide and the HR-C peptide, but also providing anattachment point itself for including but not limited to, a polymer,biologically active molecule, PEG or other chemical linker.

A description directed to a “polypeptide” applies equally to adescription of a “peptide” and vice versa. The terms “polypeptide”,“peptide”, and “protein” apply to naturally occurring amino acidpolymers as well as amino acid polymers in which one or more amino acidresidues is a non-naturally encoded amino acid. One of skill of the artwould understand techniques and modifications to proteins are applicableto polypeptides and peptides, and thus BPFIs.

The term “substantially purified” refers to BPFI that may besubstantially or essentially free of components that normally accompanyor interact with the protein as found in its naturally occurringenvironment, i.e. a native cell, or host cell in the case ofrecombinantly produced BPFI. BPFI that may be substantially free ofcellular material includes preparations of protein having less thanabout 30%, less than about 25%, less than about 20%, less than about15%, less than about 10%, less than about 5%, less than about 4%, lessthan about 3%, less than about 2%, or less than about 1% (by dry weight)of contaminating protein. When the BPFI or variant thereof isrecombinantly produced by the host cells, the protein may be present atabout 30%, about 25%, about 20%, about 15%, about 10%, about 5%, about4%, about 3%, about 2%, or about 1% or less of the dry weight of thecells. When the BPFI or variant thereof is recombinantly produced by thehost cells, the protein may be present in the culture medium at about 5g/L, about 4 g/L, about 3 g/L, about 2 g/L, about 1 g/L, about 750 mg/L,about 500 mg/L, about 250 mg/L, about 100 mg/L, about 50 mg/L, about 10mg/L, or about 1 mg/L or less of the dry weight of the cells. Thus,“substantially purified” BPFI as produced by the methods of the presentinvention may have a purity level of at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 55%, at least about 60%, at least about 65%, at least about70%, specifically, a purity level of at least about 75%, 80%, 85%, andmore specifically, a purity level of at least about 90%, a purity levelof at least about 95%, a purity level of at least about 99% or greateras determined by appropriate methods such as SDS/PAGE analysis, RP-HPLC,SEC, and capillary electrophoresis.

A “recombinant host cell” or “host cell” refers to a cell that includesan exogenous polynucleotide, regardless of the method used forinsertion, for example, direct uptake, transduction, f-mating, or othermethods known in the art to create recombinant host cells. The exogenouspolynucleotide may be maintained as a nonintegrated vector, for example,a plasmid, or alternatively, may be integrated into the host genome.

As used herein, the term “medium” or “media” includes any culturemedium, solution, solid, semi-solid, or rigid support that may supportor contain any host cell, including bacterial host cells, yeast hostcells, insect host cells, plant host cells, eukaryotic host cells,mammalian host cells, CHO cells or E. coli, and cell contents. Thus, theterm may encompass medium in which the host cell has been grown, e.g.,medium into which BPFI has been secreted, including medium either beforeor after a proliferation step. The term also may encompass buffers orreagents that contain host cell lysates, such as in the case where BPFIis produced intracellularly and the host cells are lysed or disrupted torelease BPFI.

“Reducing agent,” as used herein with respect to protein refolding, isdefined as any compound or material which maintains sulfhydryl groups inthe reduced state and reduces intra- or intermolecular disulfide bonds.Suitable reducing agents include, but are not limited to, dithiothreitol(DTT), 2-mercaptoethanol, dithioerythritol, cysteine, cysteamine(2-aminoethanethiol), and reduced glutathione. It is readily apparent tothose of ordinary skill in the art that a wide variety of reducingagents are suitable for use in the methods and compositions of thepresent invention.

“Oxidizing agent,” as used hereinwith respect to protein refolding, isdefined as any compound or material which is capable of removing anelectron from a compound being oxidized. Suitable oxidizing agentsinclude, but are not limited to, oxidized glutathione, cystine,cystamine, oxidized dithiothreitol, oxidized erythreitol, and oxygen. Itis readily apparent to those of ordinary skill in the art that a widevariety of oxidizing agents are suitable for use in the methods of thepresent invention.

“Denaturing agent” or “denaturant,” as used herein, is defined as anycompound or material which will cause a reversible unfolding of apolypeptide. The strength of a denaturing agent or denaturant will bedetermined both by the properties and the concentration of theparticular denaturing agent or denaturant. Suitable denaturing agents ordenaturants may be chaotropes, detergents, organic solvents, watermiscible solvents, phospholipids, or a combination of two or more suchagents. Suitable chaotropes include, but are not limited to, urea,guanidine, and sodium thiocyanate. Useful detergents may include, butare not limited to, strong detergents such as sodium dodecyl sulfate, orpolyoxyethylene ethers (e.g. Tween or Triton detergents), Sarkosyl, mildnon-ionic detergents (e.g., digitonin), mild cationic detergents such asN->2,3-(Dioleyoxy)-propyl-N,N,N-trimethylammonium, mild ionic detergents(e.g. sodium cholate or sodium deoxycholate) or zwitterionic detergentsincluding, but not limited to, sulfobetaines (Zwittergent),3-(3-chlolamidopropyl)dimethylammonio-1-propane sulfate (CHAPS), and3-(3-chlolamidopropyl)dimethylammonio-2-hydroxy-1-propane sulfonate(CHAPSO). Organic, water miscible solvents such as acetonitrile, loweralkanols (especially C₂-C₄ alkanols such as ethanol or isopropanol), orlower alkandiols (especially C₂-C₄ alkandiols such as ethylene-glycol)may be used as denaturants. Phospholipids useful in the presentinvention may be naturally occurring phospholipids such asphosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, andphosphatidylinositol or synthetic phospholipid derivatives or variantssuch as dihexanoylphosphatidylcholine or diheptanoylphosphatidylcholine.

“Refolding,” as used herein describes any process, reaction or methodwhich transforms disulfide bond containing polypeptides from animproperly folded or unfolded state to a native or properly foldedconformation with respect to disulfide bonds.

“Cofolding,” as used herein, refers specifically to refolding processes,reactions, or methods which employ at least two polypeptides whichinteract with each other and result in the transformation of unfolded orimproperly folded polypeptides to native, properly folded polypeptides.

As used herein, “BPFI” shall include those polypeptides and proteinsthat have at least one biological activity of a fusion inhibitor, aswell as analogs, isoforms, mimetics, fragments, hybrid proteins, fusionproteins, oligomers and multimers, homologues, glycosylation patternvariants, and muteins, thereof, regardless of the biological activity ofsame, and further regardless of the method of synthesis or manufacturethereof including, but not limited to, recombinant (whether producedfrom cDNA, genomic DNA, synthetic DNA or other form of nucleic acid),synthetic, transgenic, and gene activated methods. It is possible toobtain BPFI through the use of recombinant DNA technology, as disclosedby Maniatis, T., et al., Molecular Biology: A Laboratory Manual, ColdSpring Harbor, N.Y. (1982), and produce BPFI in host cells by methodsknown to one of ordinary skill in the art.

BPFI also include the pharmaceutically acceptable salts and prodrugs,and prodrugs of the salts, polymorphs, hydrates, solvates,biologically-active fragments, biologically active variants andstereoisomers of the naturally-occurring HR-C, HR-N, and/or anionicpeptides as well as agonist, mimetic, and antagonist variants of thenaturally-occurring HR-C, HR-N, and/or anionic peptides, and polypeptidefusions thereof. Fusions comprising additional amino acids at the aminoterminus, carboxyl terminus, or both, are encompassed by the term“BPFI.” Exemplary fusions include, but are not limited to, e.g.,methionyl BPFI in which a methionine is linked to the N-terminus of BPFIresulting from the recombinant expression of BPFI, fusions for thepurpose of purification (including, but not limited to, topoly-histidine or affinity epitopes), fusions with serum albumin bindingpeptides; fusions with serum proteins such as serum albumin; fusionswith constant regions of immunoglobulin molecules such as Fc; andfusions with fatty acids. The naturally-occurring HR-C, HR-N, andanionic peptide nucleic acid and amino acid sequences for various formsare known, as are variants such as single amino acid variants or splicevariants.

Various references disclose modification of polypeptides by polymerconjugation or glycosylation. The term BPFI includes polypeptidesconjugated to a polymer such as PEG and may be comprised of one or moreadditional derivitizations of cysteine, lysine, or other residues. Inaddition, BPFIs may comprise a linker or polymer, wherein the amino acidto which the linker or polymer is conjugated may be a non-natural aminoacid according to the present invention, or may be conjugated to anaturally encoded amino acid utilizing techniques known in the art suchas coupling to lysine or cysteine.

Polymer modification of polypeptides has been reported. U.S. Pat. No.4,904,584 discloses PEGylated lysine depleted polypeptides, wherein atleast one lysine residue has been deleted or replaced with any otheramino acid residue. WO 99/67291 discloses a process for conjugating aprotein with PEG, wherein at least one amino acid residue on the proteinis deleted and the protein is contacted with PEG under conditionssufficient to achieve conjugation to the protein. WO 99/03887 disclosesPEGylated variants of polypeptides belonging to the growth hormonesuperfamily, wherein a cysteine residue has been susbstituted with anon-essential amino acid residue located in a specified region of thepolypeptide. WO 00/26354 discloses a method of producing a glycosylatedpolypeptide variant with reduced allergenicity, which as compared to acorresponding parent polypeptide comprises at least one additionalglycosylation site. U.S. Pat. No. 5,218,092 discloses modification ofgranulocyte colony stimulating factor (G-CSF) and other polypeptides soas to introduce at least one additional carbohydrate chain as comparedto the native polypeptide. Examples of PEGylated peptides includeGW395058, a PEGylated peptide thrombopoietin receptor (TPOr) agonist (deSerres M., et al., Stem Cells. 1999; 17(4):203-9), and a PEGylatedanalogue of growth hormone releasing factor (PEG-GRP; D'Antonio M, etal. Growth Horm IGF Res. 2004 June; 14(3):226-34).

The term BPFI also includes glycosylated BPFI's, such as but not limitedto, BPFIs glycosylated at any amino acid position, N-linked or O-linkedglycosylated forms of the polypeptide. Variants containing singlenucleotide changes are also considered as biologically active variantsof BPFI. In addition, splice variants are also included. The term BPFIalso includes BPFI heterodimers, homodimers, heteromultimers, orhomomultimers of any one or more BPFI or any other polypeptide, protein,carbohydrate, polymer, small molecule, linker, ligand, or otherbiologically active molecule of any type, linked by chemical means orexpressed as a fusion protein, as well as polypeptide analoguescontaining, for example, specific deletions or other modifications yetmaintain biological activity.

The term BPFI encompasses BPFI polypeptides comprising one or more aminoacid substitutions, additions or deletions. BPFIs of the presentinvention may be comprised of modifications with one or more naturalamino acids in conjunction with one or more non-natural amino acidmodification. Exemplary substitutions in a wide variety of amino acidpositions in naturally-occurring BPFIs have been described, includingbut not limited to substitutions that modulate one or more of thebiological activities of the BPFI, such as but not limited to, increaseagonist activity, increase solubility of the polypeptide, convert thepolypeptide into an antagonist, decrease peptidase or proteasesusceptibility, etc. and are encompassed by the term BPFI.

In some embodiments, the BPFIs further comprise an addition,substitution or deletion that modulates biological activity of BPFI. Forexample, the additions, substitution or deletions may modulate one ormore properties or activities of BPFI. For example, the additions,substitutions or deletions may modulate affinity for the BPFI receptoror binding partner, modulate (including but not limited to, increases ordecreases) receptor dimerization, stabilize receptor dimers, modulatethe conformation or one or more biological activities of a bindingpartner, modulate circulating half-life, modulate therapeutic half-life,modulate stability of the polypeptide, modulate cleavage by peptidasesor proteases, modulate dose, modulate release or bio-availability,facilitate purification, or improve or alter a particular route ofadministration. Similarly, BPFIs may comprise protease cleavagesequences, reactive groups, antibody-binding domains (including but notlimited to, FLAG or poly-His) or other affinity based sequences(including but not limited to, FLAG, poly-His, GST, etc.) or linkedmolecules (including but not limited to, biotin) that improve detection(including but not limited to, GFP), purification or other traits of thepolypeptide.

The term BPFI also encompasses homodimers, heterodimers, homomultimers,and heteromultimers that are linked, including but not limited to thoselinked directly via non-naturally encoded amino acid side chains, eitherto the same or different non-naturally encoded amino acid side chains,to naturally-encoded amino acid side chains, or indirectly via a linker.Exemplary linkers including but are not limited to, small organiccompounds, water soluble polymers of a variety of lengths such aspoly(ethylene glycol) or polydextran, or polypeptides of variouslengths.

A “non-naturally encoded amino acid” refers to an amino acid that is notone of the 20 common amino acids or pyrolysine or selenocysteine. Otherterms that may be used synonymously with the term “non-naturally encodedamino acid” are “non-natural amino acid,” “unnatural amino acid,”“non-naturally-occurring amino acid,” and variously hyphenated andnon-hyphenated versions thereof. The term “non-naturally encoded aminoacid” also includes, but is not limited to, amino acids that occur bymodification (e.g. post-translational modifications) of a naturallyencoded amino acid (including but not limited to, the 20 common aminoacids or pyrolysine and selenocysteine) but are not themselves naturallyincorporated into a growing polypeptide chain by the translationcomplex. Examples of such non-naturally-occurring amino acids include,but are not limited to, N-acetylglucosaminyl-L-serine,N-acetylglucosaminyl-L-threonine, and O-phosphotyrosine.

An “amino terminus modification group” refers to any molecule that canbe attached to the amino terminus of a polypeptide. Similarly, a“carboxy terminus modification group” refers to any molecule that can beattached to the carboxy terminus of a polypeptide. Terminus modificationgroups include, but are not limited to, various water soluble polymers,peptides or proteins such as serum albumin, immunoglobulin constantregion portions such as Fc, or other moieties that increase serumhalf-life of peptides.

The terms “functional group”, “active moiety”, “activating group”,“leaving group”, “reactive site”, “chemically reactive group” and“chemically reactive moiety” are used in the art and herein to refer todistinct, definable portions or units of a molecule. The terms aresomewhat synonymous in the chemical arts and are used herein to indicatethe portions of molecules that perform some function or activity and arereactive with other molecules.

The term “linkage” or “linker” is used herein to refer to groups orbonds that normally are formed as the result of a chemical reaction andtypically are covalent linkages. Hydrolytically stable linkages meansthat the linkages are substantially stable in water and do not reactwith water at useful pH values, including but not limited to, underphysiological conditions for an extended period of time, perhaps evenindefinitely. Hydrolytically unstable or degradable linkages mean thatthe linkages are degradable in water or in aqueous solutions, includingfor example, blood. Enzymatically unstable or degradable linkages meanthat the linkage can be degraded by one or more enzymes. As understoodin the art, PEG and related polymers may include degradable linkages inthe polymer backbone or in the linker group between the polymer backboneand one or more of the terminal functional groups of the polymermolecule. For example, ester linkages formed by the reaction of PEGcarboxylic acids or activated PEG carboxylic acids with alcohol groupson a biologically active agent generally hydrolyze under physiologicalconditions to release the agent. Other hydrolytically degradablelinkages include, but are not limited to, carbonate linkages; iminelinkages resulted from reaction of an amine and an aldehyde; phosphateester linkages formed by reacting an alcohol with a phosphate group;hydrazone linkages which are reaction product of a hydrazide and analdehyde; acetal linkages that are the reaction product of an aldehydeand an alcohol; orthoester linkages that are the reaction product of aformate and an alcohol; peptide linkages formed by an amine group,including but not limited to, at an end of a polymer such as PEG, and acarboxyl group of a peptide; and oligonucleotide linkages formed by aphosphoramidite group, including but not limited to, at the end of apolymer, and a 5′ hydroxyl group of an oligonucleotide.

The term “biologically active molecule”, “biologically active moiety” or“biologically active agent” when used herein means any substance whichcan affect any physical or biochemical properties of a biologicalsystem, pathway, molecule, or interaction relating to an organism,including but not limited to, viruses, bacteria, bacteriophage,transposon, prion, insects, fungi, plants, animals, and humans. Inparticular, as used herein, biologically active molecules include, butare not limited to, any substance intended for diagnosis, cure,mitigation, treatment, or prevention of disease in humans or otheranimals, or to otherwise enhance physical or mental well-being of humansor animals. Examples of biologically active molecules include, but arenot limited to, peptides, proteins, enzymes, small molecule drugs, harddrugs, soft drugs, carbohydrates, inorganic atoms or molecules, dyes,lipids, nucleosides, radionuclides, oligonucleotides, toxins, cells,viruses, liposomes, microparticles and micelles. Classes of biologicallyactive agents that are suitable for use with the invention include, butare not limited to, drugs, prodrugs, radionuclides, imaging agents,polymers, antibiotics, fungicides, anti-viral agents, anti-inflammatoryagents, anti-tumor agents, cardiovascular agents, anti-anxiety agents,hormones, growth factors, steroidal agents, microbially derived toxins,and the like.

A “bifunctional polymer” refers to a polymer comprising two discretefunctional groups that are capable of reacting specifically with othermoieties (including but not limited to, amino acid side groups) to formcovalent or non-covalent linkages. A bifunctional linker having onefunctional group reactive with a group on a particular biologicallyactive component, and another group reactive with a group on a secondbiological component, may be used to form a conjugate that includes thefirst biologically active component, the bifunctional linker and thesecond biologically active component. Many procedures and linkermolecules for attachment of various compounds to peptides are known.See, e.g., European Patent Application No. 188,256; U.S. Pat. Nos.4,671,958, 4,659,839, 4,414,148, 4,699,784; 4,680,338; 4,569,789; and4,589,071 which are incorporated by reference herein. A“multi-functional polymer” refers to a polymer comprising two or morediscrete functional groups that are capable of reacting specificallywith other moieties (including but not limited to, amino acid sidegroups) to form covalent or non-covalent linkages. A bi-functionalpolymer or multi-functional polymer may be any desired molecular lengthor molecular weight, and may be selected to provide a particular desiredspacing or conformation between one or more molecules linked to the BPFIand its binding partner or the BPFI.

Where substituent groups are specified by their conventional chemicalformulas, written from left to right, they equally encompass thechemically identical substituents that would result from writing thestructure from right to left, for example, the structure —CH₂O— isequivalent to the structure —OCH₂—.

The term “substituents” includes but is not limited to “non-interferingsubstituents”. “Non-interfering substituents” are those groups thatyield stable compounds. Suitable non-interfering substituents orradicals include, but are not limited to, halo, C₁-C₁₀ alkyl, C₂-C₁₀alkenyl, C₂-C₁₀ alkynyl, C₁-C₁₀ alkoxy, C₁-C₁₂ aralkyl, C₁-C₁₂ alkaryl,C₃-C₁₂ cycloalkyl, C₃-C₁₂ cycloalkenyl, phenyl, substituted phenyl,toluoyl, xylenyl, biphenyl, C₂-C₁₂ alkoxyalkyl, C₂-C₁₂ alkoxyaryl,C₇-C₁₂ aryloxyalkyl, C₇-C₁₂ oxyaryl, C₁-C₆ alkylsulfinyl, C₁-C₁₀alkylsulfonyl, —(CH₂)_(m)—O—(C₁-C₁₀ alkyl) wherein m is from 1 to 8,aryl, substituted aryl, substituted alkoxy, fluoroalkyl, heterocyclicradical, substituted heterocyclic radical, nitroalkyl, —NO₂, —CN,—NRC(O)—(C₁-C₁₀ alkyl), —C(O)—(C₁-C₁₀ alkyl), C₂-C₁₀ alkyl thioalkyl,—C(O)O—(C₁-C₁₀ alkyl), —OH, —SO₂, ═S, —COOH, —NR₂, carbonyl,—C(O)—(C₁-C₁₀ alkyl)-CF3, —C(O)—CF3, —C(O)NR₂, —(C₁-C₁₀ aryl)-S—(C₆-C₁₀aryl), —C(O)—(C₁-C₁₀ aryl), —(CH₂)_(m)—O—(—(CH₂)_(m)—O—(C₁-C₁₀ alkyl)wherein each m is from 1 to 8, —C(O)NR₂, —C(S)NR₂, —SO₂NR₂, —NRC(O)NR₂,—NRC(S)NR₂, salts thereof, and the like. Each R as used herein is H,alkyl or substituted alkyl, aryl or substituted aryl, aralkyl, oralkaryl.

The term “halogen” includes fluorine, chlorine, iodine, and bromine.

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight or branched chain, or cyclichydrocarbon radical, or combination thereof, which may be fullysaturated, mono- or polyunsaturated and can include di- and multivalentradicals, having the number of carbon atoms designated (i.e. C₁-C₁₀means one to ten carbons). Examples of saturated hydrocarbon radicalsinclude, but are not limited to, groups such as methyl, ethyl, n-propyl,isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl,(cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, forexample, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. Anunsaturated alkyl group is one having one or more double bonds or triplebonds. Examples of unsaturated alkyl groups include, but are not limitedto, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl),2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl,3-butynyl, and the higher homologs and isomers. The term “alkyl,” unlessotherwise noted, is also meant to include those derivatives of alkyldefined in more detail below, such as “heteroalkyl.” Alkyl groups whichare limited to hydrocarbon groups are termed “homoalkyl”.

The term “alkylene” by itself or as part of another substituent means adivalent radical derived from an alkane, as exemplified, but notlimited, by the structures —CH₂CH₂— and —CH₂CH₂CH₂CH₂—, and furtherincludes those groups described below as “heteroalkylene.” Typically, analkyl (or alkylene) group will have from 1 to 24 carbon atoms, withthose groups having 10 or fewer carbon atoms being preferred in thepresent invention. A “lower alkyl” or “lower alkylene” is a shorterchain alkyl or alkylene group, generally having eight or fewer carbonatoms.

The terms “alkoxy,” “alkylamino” and “alkylthio” (or thioalkoxy) areused in their conventional sense, and refer to those alkyl groupsattached to the remainder of the molecule via an oxygen atom, an aminogroup, or a sulfur atom, respectively.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chain, orcyclic hydrocarbon radical, or combinations thereof, consisting of thestated number of carbon atoms and at least one heteroatom selected fromthe group consisting of O, N, Si and S, and wherein the nitrogen andsulfur atoms may optionally be oxidized and the nitrogen heteroatom mayoptionally be quaternized. The heteroatom(s) O, N and S and Si may beplaced at any interior position of the heteroalkyl group or at theposition at which the alkyl group is attached to the remainder of themolecule. Examples include, but are not limited to, —CH₂—CH₂—O—CH₃,—CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃, —CH₂—CH₂,—S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃, —CH₂—CH═N—OCH₃,and —CH═CH—N(CH₃)—CH₃. Up to two heteroatoms may be consecutive, suchas, for example, —CH₂—NH—OCH₃ and —CH₂—O—Si(CH₃)₃. Similarly, the term“heteroalkylene” by itself or as part of another substituent means adivalent radical derived from heteroalkyl, as exemplified, but notlimited by, —CH₂—CH₂—S—CH₂—CH₂— and —CH₂—S—CH₂—CH₂—NH—CH₂—. Forheteroalkylene groups, the same or different heteroatoms can also occupyeither or both of the chain termini (including but not limited to,alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino,aminooxyalkylene, and the like). Still further, for alkylene andheteroalkylene linking groups, no orientation of the linking group isimplied by the direction in which the formula of the linking group iswritten. For example, the formula —C(O)₂R′— represents both —C(O)₂R′—and —R′C(O)₂—.

The terms “cycloalkyl” and “heterocycloalkyl”, by themselves or incombination with other terms, represent, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl”, respectively. Thus, a cycloalkylor heterocycloalkyl include saturated and unsaturated ring linkages.Additionally, for heterocycloalkyl, a heteroatom can occupy the positionat which the heterocycle is attached to the remainder of the molecule.Examples of cycloalkyl include, but are not limited to, cyclopentyl,cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like.Examples of heterocycloalkyl include, but are not limited to,1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like. Additionally, the termencompasses bicyclic and tricyclic ring structures. Similarly, the term“heterocycloalkylene” by itself or as part of another substituent meansa divalent radical derived from heterocycloalkyl, and the term“cycloalkylene” by itself or as part of another substituent means adivalent radical derived from cycloalkyl.

As used herein, the term “water soluble polymer” refers to any polymerthat is soluble in aqueous solvents. Linkage of water soluble polymersto BPFI can result in changes including, but not limited to, increasedor modulated serum half-life, or increased or modulated therapeutichalf-life relative to the unmodified form, modulated immunogenicity,modulated physical association characteristics such as aggregation andmultimer formation, altered receptor binding, altered binding to one ormore binding partners, and altered receptor dimerization ormultimerization. The water soluble polymer may or may not have its ownbiological activity, and may be utilized as a linker for attaching BPFIto other substances, including but not limited to, one or more BPFIs orone or more biologically active molecules. Suitable polymers include,but are not limited to, polyethylene glycol, polyethylene glycolpropionaldehyde, mono C1-C10 alkoxy or aryloxy derivatives thereof(described in U.S. Pat. No. 5,252,714 which is incorporated by referenceherein), monomethoxy-polyethylene glycol, polyvinyl pyrrolidone,polyvinyl alcohol, polyamino acids, divinylether maleic anhydride,N-(2-Hydroxypropyl)-methacrylamide, dextran, dextran derivativesincluding dextran sulfate, polypropylene glycol, polypropyleneoxide/ethylene oxide copolymer, polyoxyethylated polyol, heparin,heparin fragments, polysaccharides, oligosaccharides, glycans, celluloseand cellulose derivatives, including but not limited to methylcelluloseand carboxymethyl cellulose, starch and starch derivatives,polypeptides, polyalkylene glycol and derivatives thereof, copolymers ofpolyalkylene glycols and derivatives thereof, polyvinyl ethyl ethers,and alpha-beta-poly[(2-hydroxyethyl)-DL-aspartamide, and the like, ormixtures thereof. Examples of such water soluble polymers include, butare not limited to, polyethylene glycol and serum albumin.

As used herein, the term “polyalkylene glycol” or “poly(alkene glycol)”refers to polyethylene glycol (poly(ethylene glycol)), polypropyleneglycol, polybutylene glycol, and derivatives thereof. The term“polyalkylene glycol” encompasses both linear and branched polymers andaverage molecular weights of between 0.1 kDa and 100 kDa. Otherexemplary embodiments are listed, for example, in commercial suppliercatalogs, such as Shearwater Corporation's catalog “Polyethylene Glycoland Derivatives for Biomedical Applications” (2001).

The term “aryl” means, unless otherwise stated, a polyunsaturated,aromatic, hydrocarbon substituent which can be a single ring or multiplerings (preferably from 1 to 3 rings) which are fused together or linkedcovalently. The term “heteroaryl” refers to aryl groups (or rings) thatcontain from one to four heteroatoms selected from N, O, and S, whereinthe nitrogen and sulfur atoms are optionally oxidized, and the nitrogenatom(s) are optionally quaternized. A heteroaryl group can be attachedto the remainder of the molecule through a heteroatom. Non-limitingexamples of aryl and heteroaryl groups include phenyl, 1-naphthyl,2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl,2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl,2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl,2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl,4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl,1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,3-quinolyl, and 6-quinolyl. Substituents for each of the above notedaryl and heteroaryl ring systems are selected from the group ofacceptable substituents described below.

For brevity, the term “aryl” when used in combination with other terms(including but not limited to, aryloxy, arylthioxy, arylalkyl) includesboth aryl and heteroaryl rings as defined above. Thus, the term“arylalkyl” is meant to include those radicals in which an aryl group isattached to an alkyl group (including but not limited to, benzyl,phenethyl, pyridylmethyl and the like) including those alkyl groups inwhich a carbon atom (including but not limited to, a methylene group)has been replaced by, for example, an oxygen atom (including but notlimited to, phenoxymethyl, 2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl,and the like).

Each of the above terms (including but not limited to, “alkyl,”“heteroalkyl,” “aryl” and “heteroaryl”) are meant to include bothsubstituted and unsubstituted forms of the indicated radical. Exemplarysubstituents for each type of radical are provided below.

Substituents for the alkyl and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) can be one or more of a variety of groups selectedfrom, but not limited to: —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′,-halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and—NO₂ in a number ranging from zero to (2 m′+1), where m′ is the totalnumber of carbon atoms in such a radical. R′, R″, R′″ and R″″ eachindependently refer to hydrogen, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, including but notlimited to, aryl substituted with 1-3 halogens, substituted orunsubstituted alkyl, alkoxy or thioalkoxy groups, or arylalkyl groups.When a compound of the invention includes more than one R group, forexample, each of the R groups is independently selected as are each R′,R″, R′″ and R″″ groups when more than one of these groups is present.When R′ and R″ are attached to the same nitrogen atom, they can becombined with the nitrogen atom to form a 5-, 6-, or 7-membered ring.For example, —NR′R″ is meant to include, but not be limited to,1-pyrrolidinyl and 4-morpholinyl. From the above discussion ofsubstituents, one of skill in the art will understand that the term“alkyl” is meant to include groups including carbon atoms bound togroups other than hydrogen groups, such as haloalkyl (including but notlimited to, —CF₃ and —CH₂CF₃) and acyl (including but not limited to,—C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and the like).

Similar to the substituents described for the alkyl radical,substituents for the aryl and heteroaryl groups are varied and areselected from, but are not limited to: halogen, —OR′, ═O, ═NR′, ═N—OR′,—NR′R″, —SR′, -halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″,—OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′,—NR—C(NR′R″R′″)═NR″″, —NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″,—NRSO₂R′, —CN and —NO₂, —R′, —N₃, —CH(Ph)₂, fluoro(C₁-C₄)alkoxy, andfluoro(C₁-C₄)alkyl, in a number ranging from zero to the total number ofopen valences on the aromatic ring system; and where R′, R″, R′″ and R″″are independently selected from hydrogen, alkyl, heteroalkyl, aryl andheteroaryl. When a compound of the invention includes more than one Rgroup, for example, each of the R groups is independently selected asare each R′, R″, R′″ and R″″ groups when more than one of these groupsis present.

As used herein, the term “modulated serum half-life” means the positiveor negative change in circulating half-life of a modified BPFI relativeto its non-modified form. Serum half-life is measured by taking bloodsamples at various time points after administration of the BPFI, anddetermining the concentration of that molecule in each sample.Correlation of the serum concentration with time allows calculation ofthe serum half-life. Increased serum half-life desirably has at leastabout two-fold, but a smaller increase may be useful, for example whereit enables a satisfactory dosing regimen or avoids a toxic effect. Insome embodiments, the increase is at least about three-fold, at leastabout five-fold, or at least about ten-fold.

The term “modulated therapeutic half-life” as used herein means thepositive or negative change in the half-life of the therapeuticallyeffective amount of BPFI, relative to its non-modified form. Therapeutichalf-life is measured by measuring pharmacokinetic and/orpharmacodynamic properties and/or therapeutic effect of the molecule atvarious time points after administration. Increased therapeutichalf-life desirably enables a particular beneficial dosing regimen, aparticular beneficial total dose, or avoids an undesired effect. In someembodiments, the increased therapeutic half-life results from increasedpotency, increased or decreased binding of the modified molecule to itstarget, increased or decreased breakdown of the molecule by enzymes suchas peptidases or proteases, or an increase or decrease in anotherparameter or mechanism of action of the non-modified molecule.

The term “isolated,” when applied to a nucleic acid or protein, denotesthat the nucleic acid or protein is substantially free of other cellularcomponents with which it is associated in the natural state. It can bein a homogeneous state. Isolated substances can be in either a dry orsemi-dry state, or in solution, including but not limited to, an aqueoussolution. Purity and homogeneity are typically determined usinganalytical chemistry techniques such as polyacrylamide gelelectrophoresis or high performance liquid chromatography. A proteinwhich is the predominant species present in a preparation issubstantially purified. In particular, an isolated gene is separatedfrom open reading frames which flank the gene and encode a protein otherthan the gene of interest. The term “purified” denotes that a nucleicacid or protein gives rise to substantially one band in anelectrophoretic gel. Particularly, it means that the nucleic acid orprotein is at least 85% pure, at least 90% pure, at least 95% pure, atleast 99% or greater pure.

The term “nucleic acid” refers to deoxyribonucleotides,deoxyribonucleosides, ribonucleosides, or ribonucleotides and polymersthereof in either single- or double-stranded form. Unless specificallylimited, the term encompasses nucleic acids containing known analoguesof natural nucleotides which have similar binding properties as thereference nucleic acid and are metabolized in a manner similar tonaturally occurring nucleotides. Unless specifically limited otherwise,the term also refers to oligonucleotide analogs including PNA(peptidonucleic acid), analogs of DNA used in antisense technology(phosphorothioates, phosphoroamidates, and the like). Unless otherwiseindicated, a particular nucleic acid sequence also implicitlyencompasses conservatively modified variants thereof (including but notlimited to, degenerate codon substitutions) and complementary sequencesas well as the sequence explicitly indicated. Specifically, degeneratecodon substitutions may be achieved by generating sequences in which thethird position of one or more selected (or all) codons is substitutedwith mixed-base and/or deoxyinosine residues (Batzer et al., NucleicAcid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608(1985); and Cassol et al. (1992); Rossolini et al., Mol. Cell. Probes8:91-98 (1994)).

The term “amino acid” refers to naturally occurring and non-naturallyoccurring amino acids, as well as amino acid analogs and amino acidmimetics that function in a manner similar to the naturally occurringamino acids. Naturally encoded amino acids are the 20 common amino acids(alanine, arginine, asparagine, aspartic acid, cysteine, glutamine,glutamic acid, glycine, histidine, isoleucine, leucine, lysine,methionine, phenylalanine, proline, serine, threonine, tryptophan,tyrosine, and valine) and pyrolysine and selenocysteine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an α carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, such as,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (such as, norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, “conservatively modified variants” refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidwhich encodes a polypeptide is implicit in each described sequence.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the invention.

The following eight groups each contain amino acids that areconservative substitutions for one another:

1) Alanine (A), Glycine (G);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5)Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6)Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S),Threonine (T); and 8) Cysteine (C), Methionine (M)

(see, e.g., Creighton, Proteins: Structures and Molecular Properties (WH Freeman & Co.; 2nd edition (December 1993)

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same. Sequences are“substantially identical” if they have a percentage of amino acidresidues or nucleotides that are the same (i.e., about 60% identity,optionally about 65%, about 70%, about 75%, about 80%, about 85%, about90%, or about 95% identity over a specified region), when compared andaligned for maximum correspondence over a comparison window, ordesignated region as measured using one of the following sequencecomparison algorithms or by manual alignment and visual inspection. Thisdefinition also refers to the complement of a test sequence. Theidentity can exist over a region that is at least about 50 amino acidsor nucleotides in length, or over a region that is 75-100 amino acids ornucleotides in length, or, where not specified, across the entiresequence of a polynucleotide or polypeptide, or less than 50 amino acidsor nucleotides in length.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well-known in the art. Optimal alignment of sequencesfor comparison can be conducted, including but not limited to, by thelocal homology algorithm of Smith and Waterman (1970) Adv. Appl. Math.2:482c, by the homology alignment algorithm of Needleman and Wunsch(1970) J Mol. Biol. 48:443, by the search for similarity method ofPearson and Lipman (1988) Proc. Nat'l. Acad. Sci. USA 85:2444, bycomputerized implementations of these algorithms (GAP, BESTFIT, FASTA,and TFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup, 575 Science Dr., Madison, Wis.), or by manual alignment andvisual inspection (see, e.g., Ausubel et al., Current Protocols inMolecular Biology (1995 supplement)).

One example of an algorithm that is suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al. (1997) Nuc. AcidsRes. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410,respectively. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information. TheBLAST algorithm parameters W, T, and X determine the sensitivity andspeed of the alignment. The BLASTN program (for nucleotide sequences)uses as defaults a wordlength (W) of 11, an expectation (E) or 10, M=5,N=−4 and a comparison of both strands. For amino acid sequences, theBLASTP program uses as defaults a wordlength of 3, and expectation (E)of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992)Proc. Natl. Acad. Sci. USA 89:10915) alignments (B) of 50, expectation(E) of 10, M=5, N=−4, and a comparison of both strands. The BLASTalgorithm is typically performed with the “low complexity” filter turnedoff.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin and Altschul (1993)Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01, and most preferably less than about 0.001.

The phrase “selectively (or specifically) hybridizes to” refers to thebinding, duplexing, or hybridizing of a molecule only to a particularnucleotide sequence under stringent hybridization conditions when thatsequence is present in a complex mixture (including but not limited to,total cellular or library DNA or RNA).

The phrase “stringent hybridization conditions” refers to conditions oflow ionic strength and high temperature as is known in the art.Typically, under stringent conditions a probe will hybridize to itstarget subsequence in a complex mixture of nucleic acid (including butnot limited to, total cellular or library DNA or RNA) but does nothybridize to other sequences in the complex mixture. Stringentconditions are sequence-dependent and will be different in differentcircumstances. Longer sequences hybridize specifically at highertemperatures. An extensive guide to the hybridization of nucleic acidsis found in Tijssen, Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Probes, “Overview of principles ofhybridization and the strategy of nucleic acid assays” (1993).Generally, stringent conditions are selected to be about 5-10° C. lowerthan the thermal melting point (T_(m)) for the specific sequence at adefined ionic strength pH. The T_(m) is the temperature (under definedionic strength, pH, and nucleic concentration) at which 50% of theprobes complementary to the target hybridize to the target sequence atequilibrium (as the target sequences are present in excess, at T_(m),50% of the probes are occupied at equilibrium). Stringent conditions maybe those in which the salt concentration is less than about 1.0 M sodiumion, typically about 0.01 to 1.0 M sodium ion concentration (or othersalts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. forshort probes (including but not limited to, 10 to 50 nucleotides) and atleast about 60° C. for long probes (including but not limited to,greater than 50 nucleotides). Stringent conditions may also be achievedwith the addition of destabilizing agents such as formamide. Forselective or specific hybridization, a positive signal may be at leasttwo times background, optionally 10 times background hybridization.Exemplary stringent hybridization conditions can be as following: 50%formamide, 5×SSC, and 1% SDS, incubating at 42° C., or 5×SSC, 1% SDS,incubating at 65° C., with wash in 0.2×SSC, and 0.1% SDS at 65° C. Suchwashes can be performed for 5, 15, 30, 60, 120, or more minutes.

As used herein, the term “eukaryote” refers to organisms belonging tothe phylogenetic domain Eucarya such as animals (including but notlimited to, mammals, insects, reptiles, birds, etc.), ciliates, plants(including but not limited to, monocots, dicots, algae, etc.), fungi,yeasts, flagellates, microsporidia, protists, etc.

As used herein, the term “non-eukaryote” refers to non-eukaryoticorganisms. For example, a non-eukaryotic organism can belong to theEubacteria (including but not limited to, E scherichia coli, Thermusthermophilus, Bacillus stearothermophilus, Pseudomonas fluorescens,Pseudomonas aeruginosa, Pseudomonas putida, etc.) phylogenetic domain,or the Archaea (including but not limited to, Methanococcus jannaschii,Methanobacterium thermoautotrophicum, Halobacterium such as Haloferaxvolcanii and Halobacterium species NRC-1, Archaeoglobus fulgidus,Pyrococcus furiosus, Pyrococcus horikoshii, Aeuropyrum pernix, etc.)phylogenetic domain.

The term “subject” as used herein, refers to an animal, preferably amammal, most preferably a human, who is the object of treatment,observation or experiment.

The term “effective amount” as used herein refers to that amount of the(modified) non-natural amino acid polypeptide being administered whichwill relieve to some extent one or more of the symptoms of the disease,condition or disorder being treated. Compositions containing the(modified) non-natural amino acid polypeptide described herein can beadministered for prophylactic, enhancing, and/or therapeutic treatments.

The terms “enhance” or “enhancing” means to increase or prolong eitherin potency or duration a desired effect. Thus, in regard to enhancingthe effect of therapeutic agents, the term “enhancing” refers to theability to increase or prolong, either in potency or duration, theeffect of other therapeutic agents on a system. An “enhancing-effectiveamount,” as used herein, refers to an amount adequate to enhance theeffect of another therapeutic agent in a desired system. When used in apatient, amounts effective for this use will depend on the severity andcourse of the disease, disorder or condition, previous therapy, thepatient's health status and response to the drugs, and the judgment ofthe treating physician.

The term “modified,” as used herein refers to any changes made to agiven polypeptide, such as changes to the length of the polypeptide, theamino acid sequence, amino acid composition, chemical structure,co-translational modification, or post-translational modification of apolypeptide. The form “(modified)” term means that the polypeptidesbeing discussed are optionally modified, that is, the polypeptides underdiscussion can be modified or unmodified.

The term “post-translationally modified” refers to any modification of anatural or non-natural amino acid that occurs to such an amino acidafter it has been incorporated into a polypeptide chain. The termencompasses, by way of example only, co-translational in vivomodifications, co-translational in vitro modifications (such as in acell-free translation system), post-translational in vivo modifications,and post-translational in vitro modifications.

In prophylactic applications, compositions containing the (modified)non-natural amino acid polypeptide are administered to a patientsusceptible to or otherwise at risk of a particular disease, disorder orcondition. Such an amount is defined to be a “prophylactically effectiveamount.” In this use, the precise amounts also depend on the patient'sstate of health, weight, and the like. It is considered well within theskill of the art for one to determine such prophylactically effectiveamounts by routine experimentation (e.g., a dose escalation clinicaltrial).

The term “protected” refers to the presence of a “protecting group” ormoiety that prevents reaction of the chemically reactive functionalgroup under certain reaction conditions. The protecting group will varydepending on the type of chemically reactive group being protected. Forexample, if the chemically reactive group is an amine or a hydrazide,the protecting group can be selected from the group oftert-butyloxycarbonyl (t-Boc) and 9-fluorenylmethoxycarbonyl (Fmoc). Ifthe chemically reactive group is a thiol, the protecting group can beorthopyridyldisulfide. If the chemically reactive group is a carboxylicacid, such as butanoic or propionic acid, or a hydroxyl group, theprotecting group can be benzyl or an alkyl group such as methyl, ethyl,or tert-butyl. Other protecting groups known in the art may also be usedin or with the methods and compositions described herein.

By way of example only, blocking/protecting groups may be selected from:

Other protecting groups are described in Greene and Wuts, ProtectiveGroups in Organic Synthesis, 3rd Ed., John Wiley & Sons, New York, N.Y.,1999, which is incorporated herein by reference in its entirety.

In therapeutic applications, compositions containing the (modified)non-natural amino acid polypeptide are administered to a patient alreadysuffering from a disease, condition or disorder, in an amount sufficientto cure or at least partially arrest the symptoms of the disease,disorder or condition. Such an amount is defined to be a“therapeutically effective amount,” and will depend on the severity andcourse of the disease, disorder or condition, previous therapy, thepatient's health status and response to the drugs, and the judgment ofthe treating physician. It is considered well within the skill of theart for one to determine such therapeutically effective amounts byroutine experimentation (e.g., a dose escalation clinical trial).

The term “treating” is used to refer to either prophylactic and/ortherapeutic treatments.

Unless otherwise indicated, conventional methods of mass spectroscopy,NMR, HPLC, protein chemistry, biochemistry, recombinant DNA techniquesand pharmacology, within the skill of the art are employed.

DETAILED DESCRIPTION I. Introduction

Non-limiting examples of BPFIs or fragments thereof that may be usefulin the present invention include the following. It is to be understoodthat other variants, analogs, fragments, and/or analog fragments thatretain some or all of the activity of the particular BPFI or any proteinmay also be useful in embodiments of the present invention.

Representative non-limiting classes of polypeptides useful in thepresent invention include: HR-C, HR-N, and anionic peptides.

Paramyxoviruses and lentiviruses are important agents of clinical andveterinary disease. These viruses include important human pathogens suchas respiratory syncytial virus (RSV), parainfluenza viruses, measles,mumps, HIV-1 and HIV-2, and veterinary pathogens such as bovine RSV,turkey rhinotracheitis virus, Newcastle's disease virus, rinderpestvirus, canine distemper virus, the new morbilliviruses described inseals and horses, and simian immunodeficiency virus (SIV).

The major cause of serious lower respiratory tract illness in infantsand immunosuppressed individuals is a paramyxovirus known as respiratorysyncytial virus (RSV). Worldwide, RSV causes 65 million infections and 1million deaths annually. The greatest incidence of disease from RSVinfection is from 6 weeks to 6 months of age, with approximately 90,000children hospitalized each year in the United States with infectionscaused by RSV. 4500 of those children die. Exaggerated RSV IgE responseduring RSV bronchiolitis in infancy has also been associated with thewidespread problem of recurrent wheezing in early childhood.

Reinfections with RSV are more frequent than with most other viruses ofthe respiratory tract. Serious disease is usually associated with thefirst or second infection. Although disease severity declines withrepeated infection, previous infection with RSV does not prevent illnessin subsequent infections. Immunity is apparently incomplete. Live virusvaccines have generally proven to be inadequately immunogenic by thetime they have been attenuated to a sufficient level to produce noclinical illness. A formalin-inactivated vaccine developed in the 1960snot only failed to produce a protective response against the virus, butinduced exacerbated disease in vaccinated children during a subsequentepidemic, and some attenuated RSV strains have the potential to revertto virulence after human passage. Vaccine development has therefore beenapproached cautiously, although efforts to prevent RSV disease ininfants and young children have continued to target active immunizationwith an inactivated vaccine, a live attenuated virus vaccine, or asubunit vaccine, and passive immunization of the fetus by activeimmunization of the mother with a human monoclonal RSV antibody orhyperimmune RSV immune globulin.

High-risk infants are treated with immunoglobulin (IG) to protectagainst RSV, but intravenous RSV IG is very expensive and administrationrequires a monthly infusion lasting 7 hours or more to maintainacceptable antibody titers.

Currently, Synagis (palivizumab) or drugs like ribavirin areadministered to patient populations.

U.S. Pat. No. 6,814,968, which is incorporated by reference herein,describes the use of isolated peptides, peptidomimetics, and antibodieswhich bind to the viral fusion protein binding domain of the RhoAprotein or the RhoA binding domain of a viral fusion protein ininhibiting infection in susceptible cells, in vitro and in vivo. Amongthe viruses described are the Paramyxovirus respiratory syncytial virus(RSV) and the Lentivirus human immunodeficiency virus (HIV).

Pastey et al. in Nature Medicine 2000 January; 6(1):35-40 and J. ofVirology 1999; 73(9):7262-7270 describe studies investigating theintereaction between the F protein of RSV (fusion protein) and theGTPase RhoA, and the effects of RhoA peptides on syncytium formulationby RSV and para-influenza virus type 3.

Budge et al. in J. of Antimicrobial Chemotherapy 2004; 54:299-302 and inJ. of Virology 2004; 78(10):5015-5022 describe peptides derived fromGTPase RhoA and their anti-viral activity. The inhibition of viralinfectivity and of viral attachment were measured for a set ofmolecules. In particular, the net negative charge of a peptide derivedfrom amino acids 77-95 of RhoA and intermolecular disulfide bonds of atruncated version of the peptide (amino acids 80-94) describe were shownto be critical in anti-RSV activity. Polyanionic molecules greater than5 kDa have been shown to inhibit enveloped viruses. Such moleculesinclude, but are not limited to, soluble heparin, dextran sulfate,negatively charged proteins, and synthetic polyanionic polymers. Budgeet al. suggest that the anti-viral activity is not due to inhibition ofthe RSV F protein-GTPase RhoA interaction. Lambert et al. in PNAS 119693:2186-2191 describe the use of peptides from RSV that were analogousto DP-178 and DP-107 as viral fusion inhibitors.

T-20 inhibits entry of HIV into cells by acting as a viral fusioninhibitor. The fusion process of HIV is well characterized. HIV binds toCD4 receptor via gp120, and upon binding to its receptor, gp120 goesthrough a series of conformational changes that allows it to bind to itscoreceptors, CCR5 or CXCR4. After binding to both receptor andcoreceptor, gp120 exposes gp41 to begin the fusion process. gp41 has tworegions named heptad repeat 1 and 2 (HR1 and 2). The extracellulardomain identified as HR1 is an α-helical region which is theamino-terminal of a proposed zipper domain. HR1 comes together with HR2of gp41 to form a hairpin. The structure that it is formed is a 6-helixbundle that places the HIV envelope in the proximity of the cellularmembrane causing fusion between the two menbranes. T-20 prevents theconformational changes necessary for viral fusion by binding the firstheptad-repeat (HR1) of the gp41 transmembrane glycoprotein. Thus, theformation of the 6-helix bundle is blocked by T-20's binding to the HR1region of gp41. The DP107 and DP178 domains (i.e., the HR1 and HR2domains) of the HIV gp41 protein non-covalently complex with each other,and their interaction is required for the normal infectivity of thevirus. Compounds that disrupt the interaction between DP107 and DP178,and/or between DP107-like and DP178-like peptides are antifusogenic,including antiviral.

DP-178 acts as a potent inhibitor of HIV-1 mediated CD-4⁺ cell-cellfusion (i.e., syncytial formation) and infection of CD-4⁺ cells bycell-free virus. Such anti-retroviral activity includes, but is notlimited to, the inhibition of HIV transmission to uninfected CD-4⁺cells. DP-178 act at low concentrations, and it has been proven that itis non-toxic in in vitro studies and in animals. The amino acidconservation within the DP-178-corresponding regions of HIV-1 and HIV-2has been described.

Potential uses for DP-178 peptides are described in U.S. Pat. Nos.5,464,933 and 6,133,418, as well as U.S. Pat. Nos. 6,750,008 and6,824,783, all of which are incorporated by reference herein, for use ininhibition of fusion events associated with HIV transmission.

Portions, homologs, and analogs of DP178 and DP-107 as well asmodulators of DP178/DP107, DP178-like/DP107-like or HR1/HR2 interactionshave been investigated that show antiviral activity, and/or showanti-membrane fusion capability, or an ability to modulate intracellularprocesses involving coiled-coil peptide structures in retroviruses otherthan HIV-1 and nonretroviral viruses. Viruses in such studies include,simian immunodeficiency virus (U.S. Pat. No. 6,017,536), respiratorysynctial virus (U.S. Pat. Nos. 6,228,983; 6,440,656; 6,479,055;6,623,741), Epstein-Barr virus (U.S. Pat. Nos. 6,093,794; 6,518,013),parainfluenza virus (U.S. Pat. No. 6,333,395), influenza virus (U.S.Pat. Nos. 6,068,973; 6,060,065), and measles virus (U.S. Pat. No.6,013,263). All of which are incorporated by reference herein.

A commercially available form of DP-178 is Fuzeon® (enfuvirtide, RocheLaboratories Inc. and Trimeris, Inc.). Fuzeon® has an acetylated Nterminus and a carboxamide as the C-terminus, and is described by thefollowing primary amino acid sequence:CH₃CO-YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF-NH₂. It is used incombination with other antivirals in HIV-1 patients that show HIV-1replication despite ongoing antiretroviral therapy.

U.S. Pat. Nos. 5,464,933 and 6,824,783, which are incorporated byreference herein, describes DP-178, DP-178 fragments, analogs, andhomologs, including, but not limited to, molecules with amino andcarboxy terminal truncations, substitutions, insertions, deletions,additions, or macromolecular carrier groups as well as DP-178 moleculeswith chemical groups such as hydrophobic groups present at their aminoand/or carboxy termini. Additional variants, include but are not limitedto, those described in U.S. Pat. No. 6,830,893 and the derivatives ofDP-178 disclosed in U.S. Pat. No. 6,861,059. A set of T-20 hybridpolypeptides are described in U.S. Pat. Nos. 6,656,906, 6,562,787,6,348,568 and 6,258,782, and a DP-178-toxin fusion is described in U.S.Pat. No. 6,627,197.

HAART (Highly Active Anti-Retroviral Therapy) is the standard of therapyfor HIV which combines drugs from a few classes of antiretroviral agentsto reduce viral loads. U.S. Pat. No. 6,861,059, which is incorporated byreference herein, discloses methods of treating HIV-1 infection orinhibiting HIV-1 replication employing DP-178 or DP-107 or derivativesthereof, in combination with at least one other antiviral therapeuticagent such as a reverse transcriptase inhibitor (e.g. AZT, ddI, ddC,ddA, d4T, 3TC, or other dideoxynucleotides or dideoxyfluoronucleosides)or an inhibitor of HIV-1 protease (e.g. indinavir; ritonavir). Otherantivirals include cytokines (e.g., rIFNα, rIFNβ, rIFNγ), inhibitors ofviral mRNA capping (e.g. ribavirin), inhibitors of HIV protease (e.g.ABT-538 and MK-639), amphotericin B as a lipid-binding molecule withanti-HIV activity, and castanospermine as an inhibitor of glycoproteinprocessing. Potential combination therapies of other anti-viral agents,including but not limited to, reverse transcriptase inhibitors,integrase inhibitors, protease inhibitors, cytokine antagonists, andchemokine receptor modulators with T-20 are described in a number ofreferences including U.S. Pat. Nos. 6,855,724; 6,844,340; 6,841,558;6,833,457; 6,825,210; 6,811,780; 6,809,109; 6,806,265; 6,768,007;6,750,230; 6,706,706; 6,696,494; 6,673,821; 6,673,791; 6,667,314;6,642,237; 6,599,911; 6,596,729; 6,593,346; 6,589,962; 6,586,430;6,541,515; 6,538,002; 6,531,484; 6,511,994; 6,506,777; 6,500,844;6,498,161; 6,472,410; 6,432,981; 6,410,726; 6,399,619; 6,395,743;6,358,979; 6,265,434; 6,248,755; 6,245,806; and 6,172,110.

Potential delivery systems for DP-178 include, but are not limited tothose described in U.S. Pat. Nos. 6,844,324 and 6,706,892. In addition,a process for producing T-20 in inclusion bodies was described in U.S.Pat. No. 6,858,410.

Antigenic polypeptides, which can elicit an enhanced immune response,enhance an immune response and or cause an immunizingly effectiveresponse to diseases and/or disease causing agents including, but notlimited to, respiratory syncytial virus and human immunodeficiency virus(HIV).

The present invention overcomes the problems associated with deliveringa BPFI that has a short plasma half-life. The compounds of the presentinvention encompass BPFIs fused to another protein with a longcirculating half-life such as the Fc portion of an immunoglobulin oralbumin.

Several stages of the HIV life cycle have been considered targets fortherapeutic intervention (Mitsuya, H. et al., 1991, FASEB J.5:2369-2381). Intervention could potentially inhibit the binding of HIVto cell membranes, the reverse transcription of HIV RNA genome into DNA,or the exit of the virus from the host cell and infection of newcellular targets.

Attempts are being made to develop drugs which can inhibit viral entryinto the cell, the earliest stage of HIV infection. T-20 acts as aninhibitor of HIV-1 fusion to CD4⁺ cells, targeting HIV with a differentmechanism than other antiviral therapeutics. U.S. Pat. No. 6,861,059discloses methods of treating HIV-1 infection or inhibiting HIV-1replication employing DP-178 or DP-107 or derivatives thereof, incombination with at least one other antiviral therapeutic agent such asa reverse transcriptase inhibitor (e.g. AZT, ddI, ddC, ddA, d4T, 3TC, orother dideoxynucleotides or dideoxyfluoronucleosides) or an inhibitor ofHIV-1 protease (e.g. indinavir; ritonavir). Other antivirals includecytokines (e.g., rIFNα, rIFNβ, rIFNγ), inhibitors of viral mRNA capping(e.g. ribavirin), inhibitors of HIV protease (e.g. ABT-538 and MK-639),amphotericin B as a lipid-binding molecule with anti-HIV activity, andcastanospermine as an inhibitor of glycoprotein processing.

Compounds of the present invention include heterologous fusion proteinscomprising a first polypeptide with a N-terminus and a C-terminus fusedto a second polypeptide with a N-terminus and a C-terminus wherein thefirst polypeptide is a BPFI such as anionic peptide, HR-C or HR-N, andthe second polypeptide is selected from the group consisting of a) humanalbumin; b) human albumin analogs; and c) fragments of human albumin,and wherein the C-terminus of the first polypeptide is fused to theN-terminus of the second polypeptide.

Compounds of the present invention also include a heterologous fusionprotein comprising a first polypeptide with a N-terminus and aC-terminus fused to a second polypeptide with a N-terminus and aC-terminus wherein the first polypeptide is a BPFI such as an anionicpeptide, HR-C or HR-N, and the second polypeptide is selected from thegroup consisting of a) human albumin; b) human albumin analogs; and c)fragments of human albumin, and wherein the first polypeptide is fusedto the second polypeptide via a linker, peptide linker, prodrug linker,or water soluble polymer. The peptide linker may be selected from thegroup consisting of: a) a glycine rich peptide; b) a peptide having thesequence [Gly-Gly-Gly-Gly-Ser]_(n) where n is 1, 2, 3, 4, 5, 6, or more;and c) a peptide having the sequence [Gly-Gly-Gly-Gly-Ser]₃.

Additional compounds of the present invention include a heterologousfusion protein comprising a first polypeptide with an N-terminus and aC-terminus fused to a second polypeptide with a N-terminus and aC-terminus wherein the first polypeptide is a BPFI such as a anionicpeptide, HR-C or HR-N, and the second polypeptide is selected from thegroup consisting of: a) the Fc portion of an immunoglobulin; b) ananalog of the Fc portion of an immunoglobulin; and c) fragments of theFc portion of an immunoglobulin, and wherein the C-terminus of the firstpolypeptide is fused to the N-terminus of the second polypeptide. TheBPFI such as the anionic peptide, HR-C or HR-N, may be fused to thesecond polypeptide via a peptide linker prodrug linker, or water solublepolymer. The peptide linker may be selected from the group consistingof: a) a glycine rich peptide; b) a peptide having the sequence[Gly-Gly-Gly-Gly-Ser]_(n) where n is 1, 2, 3, 4, 5, 6, or more; and c) apeptide having the sequence [Gly-Gly-Gly-Gly-Ser]₃.

The anionic peptide, HR-C or HR-N, that is part of the heterologousfusion protein may have multiple amino acid substitutions, and may havemore than 6, 5, 4, 3, 2, or 1 amino acids that differ from the nativeform of the molecules.

The present invention also includes polynucleotides encoding theheterologous fusion proteins described herein, vectors comprising thesepolynucleotides and host cells transfected or transformed with thevectors described herein. Also included is a process for producing aheterologous fusion protein comprising the steps of transcribing andtranslating a polynucleotide described herein under conditions whereinthe heterologous fusion protein is expressed in detectable amounts.

BPFI molecules comprising at least one unnatural amino acid are providedin the invention. In certain embodiments of the invention, the BPFI withat least one unnatural amino acid includes at least onepost-translational modification. In one embodiment, the at least onepost-translational modification comprises attachment of a moleculeincluding but not limited to, a label, a dye, a polymer, a water-solublepolymer, a derivative of polyethylene glycol, a photocrosslinker, aradionuclide, a cytotoxic compound, a drug, an affinity label, aphotoaffinity label, a reactive compound, a resin, a second protein orpolypeptide or polypeptide analog, an antibody or antibody fragment, ametal chelator, a cofactor, a fatty acid, a carbohydrate, apolynucleotide, a DNA, a RNA, an antisense polynucleotide, awater-soluble dendrimer, a cyclodextrin, an inhibitory ribonucleic acid,a biomaterial, a nanoparticle, a spin label, a fluorophore, ametal-containing moiety, a radioactive moiety, a novel functional group,a group that covalently or noncovalently interacts with other molecules,a photocaged moiety, a photoisomerizable moiety, biotin, a derivative ofbiotin, a biotin analogue, a moiety incorporating a heavy atom, achemically cleavable group, a photocleavable group, an elongated sidechain, a carbon-linked sugar, a redox-active agent, an amino thioacid, atoxic moiety, an isotopically labeled moiety, a biophysical probe, aphosphorescent group, a chemiluminescent group, an electron dense group,a magnetic group, an intercalating group, a chromophore, an energytransfer agent, a biologically active agent, a detectable label, a smallmolecule, or any combination of the above or any other desirablecompound or substance, comprising a second reactive group to at leastone unnatural amino acid comprising a first reactive group utilizingchemistry methodology that is known to one of ordinary skill in the artto be suitable for the particular reactive groups. For example, thefirst reactive group is an alkynyl moiety (including but not limited to,in the unnatural amino acid p-propargyloxyphenylalanine, where thepropargyl group is also sometimes referred to as an acetylene moiety)and the second reactive group is an azido moiety, and [3+2]cycloaddition chemistry methodologies are utilized. In another example,the first reactive group is the azido moiety (including but not limitedto, in the unnatural amino acid p-azido-L-phenylalanine) and the secondreactive group is the alkynyl moiety. In certain embodiments of themodified BPFI of the present invention, at least one unnatural aminoacid (including but not limited to, unnatural amino acid containing aketo functional group) comprising at least one post-translationalmodification, is used where the at least one post-translationalmodification comprises a saccharide moiety. In certain embodiments, thepost-translational modification is made in vivo in a eukaryotic cell orin a non-eukaryotic cell.

In certain embodiments, the protein includes at least onepost-translational modification that is made in vivo by one host cell,where the post-translational modification is not normally made byanother host cell type. In certain embodiments, the protein includes atleast one post-translational modification that is made in vivo by aeukaryotic cell, where the post-translational modification is notnormally made by a non-eukaryotic cell. Examples of post-translationalmodifications include, but are not limited to, acetylation, acylation,lipid-modification, palmitoylation, palmitate addition, phosphorylation,glycolipid-linkage modification, and the like. In one embodiment, thepost-translational modification comprises attachment of anoligosaccharide to an asparagine by a GlcNAc-asparagine linkage(including but not limited to, where the oligosaccharide comprises(GlcNAc-Man)₂-Man-GlcNAc-GlcNAc, and the like). In another embodiment,the post-translational modification comprises attachment of anoligosaccharide (including but not limited to, Gal-GalNAc, Gal-GlcNAc,etc.) to a serine or threonine by a GalNAc-serine, a GalNAc-threonine, aGlcNAc-serine, or a GlcNAc-threonine linkage. In certain embodiments, aprotein or polypeptide of the invention can comprise a secretion orlocalization sequence or peptide, an epitope tag, a FLAG tag, apolyhistidine tag, a GST fusion, and/or the like. Examples of tags orlinkers that may be used in the invention include, but are not limitedto, a polypeptide, a polymer, an affinity tag, an antigen, a detectiontag, an imaging tag, a member of a multiple-member binding complex, anda radio-isotope tag. Examples of affinity tags and detection tagsinclude, but are not limited to, a poly-His tag, biotin, avidin, proteinA, protein G, and an antigen including but not limited to, animmunoglobulin epitope. Examples of imaging tags include, but are notlimited to, a metal, a radionuclide, and a magnetic molecule. Examplesof multiple-member binding complex tags include, but are not limited to,streptavidin, avidin, biotin, protein A, and protein G.

The term “localization peptide” includes, but is not limited to,examples of secretion signal sequences. Examples of secretion signalsequences include, but are not limited to, a prokaryotic secretionsignal sequence, a eukaryotic secretion signal sequence, an eukaryoticsecretion signal sequence 5′-optimized for bacterial expression, a novelsecretion signal sequence, pectate lyase secretion signal sequence, OmpA secretion signal sequence, and a phage secretion signal sequence.Examples of secretion signal sequences, include, but are not limited to,STII (prokaryotic), Fd GIII and M13 (phage), Bgl2 (yeast), and thesignal sequence bla derived from a transposon. Secretion signalsequences include, but are not limited to, a bacterial secretion signalsequence, a yeast secretion signal sequence, an insect signal secretionsequence, a mammalian secretion signal sequence, and a unique secretionsignal sequence. Another example of a “localization sequence” includes,but it not limited to, a TrpLE sequence.

The protein or polypeptide of interest can contain at least one, atleast two, at least three, at least four, at least five, at least six,at least seven, at least eight, at least nine, or ten or more unnaturalamino acids. The unnatural amino acids can be the same or different, forexample, there can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more differentsites in the protein that comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or moredifferent unnatural amino acids. In certain embodiments, at least one,but fewer than all, of a particular amino acid present in a naturallyoccurring version of the protein is substituted with an unnatural aminoacid.

Any BPFI or fragment thereof with therapeutic activity may be used inthis invention. Numerous examples of BPFIs that may be used in thisinvention have been provided. However, the lists provided are notexhaustive and in no way limit the number or type of BPFIs that may beused in this invention. Thus, any BPFI and/or fragments produced fromany BPFI including novel BPFIs may be modified according to the presentinvention, and used therapeutically.

The present invention provides methods and compositions based on BPFIscomprising at least one non-naturally encoded amino acid. Introductionof at least one non-naturally encoded amino acid into BPFI can allow forthe application of conjugation chemistries that involve specificchemical reactions, including, but not limited to, with one or morenon-naturally encoded amino acids while not reacting with the commonlyoccurring 20 amino acids. In some embodiments, the BPFI, such as anionicpeptide, HR-C or HR-N, comprising the non-naturally encoded amino acidis linked to a water soluble polymer, such as polyethylene glycol (PEG),via the side chain of the non-naturally encoded amino acid. Thisinvention provides a highly efficient method for the selectivemodification of proteins with PEG derivatives, which involves theselective incorporation of non-genetically encoded amino acids,including but not limited to, those amino acids containing functionalgroups or substituents not found in the 20 naturally incorporated aminoacids, including but not limited to a ketone, an azide or acetylenemoiety, into proteins in response to a selector codon and the subsequentmodification of those amino acids with a suitably reactive PEGderivative. Once incorporated, the amino acid side chains can then bemodified by utilizing chemistry methodologies known to those of ordinaryskill in the art to be suitable for the particular functional groups orsubstituents present in the naturally encoded amino acid. Knownchemistry methodologies of a wide variety are suitable for use in thepresent invention to incorporate a water soluble polymer into theprotein. Such methodologies include but are not limited to a Huisgen[3+2] cycloaddition reaction (see, e.g., Padwa, A. in ComprehensiveOrganic Synthesis, Vol. 4, (1991) Ed. Trost, B. M., Pergamon, Oxford, p.1069-1109; and, Huisgen, R. in 1,3-Dipolar Cycloaddition Chemistry,(1984) Ed. Padwa, A., Wiley, New York, p. 1-176) with, including but notlimited to, acetylene or azide derivatives, respectively.

Because the Huisgen [3+2] cycloaddition method involves a cycloadditionrather than a nucleophilic substitution reaction, proteins can bemodified with extremely high selectivity. The reaction can be carriedout at room temperature in aqueous conditions with excellentregioselectivity (1,4>1,5) by the addition of catalytic amounts of Cu(1)salts to the reaction mixture. See, e.g., Tornoe, et al., (2002) J. Org.Chem. 67:3057-3064; and, Rostovtsev, et al., (2002) Angew. Chem. Int.Ed. 41:2596-2599; and WO 03/101972. A molecule that can be added to aprotein of the invention through a [3+2] cycloaddition includesvirtually any molecule with a suitable functional group or substituentincluding but not limited to an azido or acetylene derivative. Thesemolecules can be added to an unnatural amino acid with an acetylenegroup, including but not limited to, p-propargyloxyphenylalanine, orazido group, including but not limited to p-azido-phenylalanine,respectively.

The five-membered ring that results from the Huisgen [3+2] cycloadditionis not generally reversible in reducing environments and is stableagainst hydrolysis for extended periods in aqueous environments.Consequently, the physical and chemical characteristics of a widevariety of substances can be modified under demanding aqueous conditionswith the active PEG derivatives of the present invention. Even moreimportant, because the azide and acetylene moieties are specific for oneanother (and do not, for example, react with any of the 20 common,genetically-encoded amino acids), proteins can be modified in one ormore specific sites with extremely high selectivity.

The invention also provides water soluble and hydrolytically stablederivatives of PEG derivatives and related hydrophilic polymers havingone or more acetylene or azide moieties. The PEG polymer derivativesthat contain acetylene moieties are highly selective for coupling withazide moieties that have been introduced selectively into proteins inresponse to a selector codon. Similarly, PEG polymer derivatives thatcontain azide moieties are highly selective for coupling with acetylenemoieties that have been introduced selectively into proteins in responseto a selector codon.

More specifically, the azide moieties comprise, but are not limited to,alkyl azides, aryl azides and derivatives of these azides. Thederivatives of the alkyl and aryl azides can include other substituentsso long as the acetylene-specific reactivity is maintained. Theacetylene moieties comprise alkyl and aryl acetylenes and derivatives ofeach. The derivatives of the alkyl and aryl acetylenes can include othersubstituents so long as the azide-specific reactivity is maintained.

The present invention provides conjugates of substances having a widevariety of functional groups, substituents or moieties, with othersubstances including but not limited to a label; a dye; a polymer; awater-soluble polymer; a derivative of polyethylene glycol; aphotocrosslinker; a radionuclide; a cytotoxic compound; a drug; anaffinity label; a photoaffinity label; a reactive compound; a resin; asecond protein or polypeptide or polypeptide analog; an antibody orantibody fragment; a metal chelator; a cofactor; a fatty acid; acarbohydrate; a polynucleotide; a DNA; a RNA; an antisensepolynucleotide; a water-soluble dendrimer; a cyclodextrin; an inhibitoryribonucleic acid; a biomaterial; a nanoparticle; a spin label; afluorophore, a metal-containing moiety; a radioactive moiety; a novelfunctional group; a group that covalently or noncovalently interactswith other molecules; a photocaged moiety; a photoisomerizable moiety;biotin; a derivative of biotin; a biotin analogue; a moietyincorporating a heavy atom; a chemically cleavable group; aphotocleavable group; an elongated side chain; a carbon-linked sugar; aredox-active agent; an amino thioacid; a toxic moiety; an isotopicallylabeled moiety; a biophysical probe; a phosphorescent group; achemiluminescent group; an electron dense group; a magnetic group; anintercalating group; a chromophore; an energy transfer agent; abiologically active agent; a detectable label; a small molecule; or anycombination of the above, or any other desirable compound or substance).The present invention also includes conjugates of substances havingazide or acetylene moieties with PEG polymer derivatives having thecorresponding acetylene or azide moieties. For example, a PEG polymercontaining an azide moiety can be coupled to a biologically activemolecule at a position in the protein that contains a non-geneticallyencoded amino acid bearing an acetylene functionality. The linkage bywhich the PEG and the biologically active molecule are coupled includesbut is not limited to the Huisgen [3+2] cycloaddition product.

It is well established in the art that PEG can be used to modify thesurfaces of biomaterials (see, e.g., U.S. Pat. No. 6,610,281; Mehvar,R., J. Pharm Pharm Sci., 3(1):125-136 (2000) which are incorporated byreference herein). The invention also includes biomaterials comprising asurface having one or more reactive azide or acetylene sites and one ormore of the azide- or acetylene-containing polymers of the inventioncoupled to the surface via the Huisgen [3+2] cycloaddition linkage.Biomaterials and other substances can also be coupled to the azide- oracetylene-activated polymer derivatives through a linkage other than theazide or acetylene linkage, such as through a linkage comprising acarboxylic acid, amine, alcohol or thiol moiety, to leave the azide oracetylene moiety available for subsequent reactions.

The invention includes a method of synthesizing the azide- andacetylene-containing polymers of the invention. In the case of theazide-containing PEG derivative, the azide can be bonded directly to acarbon atom of the polymer. Alternatively, the azide-containing PEGderivative can be prepared by attaching a linking agent that has theazide moiety at one terminus to a conventional activated polymer so thatthe resulting polymer has the azide moiety at its terminus. In the caseof the acetylene-containing PEG derivative, the acetylene can be bondeddirectly to a carbon atom of the polymer. Alternatively, theacetylene-containing PEG derivative can be prepared by attaching alinking agent that has the acetylene moiety at one terminus to aconventional activated polymer so that the resulting polymer has theacetylene moiety at its terminus.

More specifically, in the case of the azide-containing PEG derivative, awater soluble polymer having at least one active hydroxyl moietyundergoes a reaction to produce a substituted polymer having a morereactive moiety, such as a mesylate, tresylate, tosylate or halogenleaving group, thereon. The preparation and use of PEG derivativescontaining sulfonyl acid halides, halogen atoms and other leaving groupsare well known to the skilled artisan. The resulting substituted polymerthen undergoes a reaction to substitute for the more reactive moiety anazide moiety at the terminus of the polymer. Alternatively, a watersoluble polymer having at least one active nucleophilic or electrophilicmoiety undergoes a reaction with a linking agent that has an azide atone terminus so that a covalent bond is formed between the PEG polymerand the linking agent and the azide moiety is positioned at the terminusof the polymer. Nucleophilic and electrophilic moieties, includingamines, thiols, hydrazides, hydrazines, alcohols, carboxylates,aldehydes, ketones, thioesters and the like, are well known to theskilled artisan.

More specifically, in the case of the acetylene-containing PEGderivative, a water soluble polymer having at least one active hydroxylmoiety undergoes a reaction to displace a halogen or other activatedleaving group from a precursor that contains an acetylene moiety.Alternatively, a water soluble polymer having at least one activenucleophilic or electrophilic moiety undergoes a reaction with a linkingagent that has an acetylene at one terminus so that a covalent bond isformed between the PEG polymer and the linking agent and the acetylenemoiety is positioned at the terminus of the polymer. The use of halogenmoieties, activated leaving group, nucleophilic and electrophilicmoieties in the context of organic synthesis and the preparation and useof PEG derivatives is well established to practitioners in the art.

The invention also provides a method for the selective modification ofproteins to add other substances to the modified protein, including butnot limited to water soluble polymers such as PEG and PEG derivativescontaining an azide or acetylene moiety. The azide- andacetylene-containing PEG derivatives can be used to modify theproperties of surfaces and molecules where biocompatibility, stability,solubility and lack of immunogenicity are important, while at the sametime providing a more selective means of attaching the PEG derivativesto proteins than was previously known in the art.

II. Peptides and Polypeptides

BPFIs that may be made utilizing the methods of the present inventionmay be any combination of amino acids, whether naturally occurring ornon-naturally encoded, of any length or sequence. The only requirementis for at least one of the amino acids in the BPFI chain to be anon-naturally encoded amino acid. If a polypeptide is madebiosynthetically, then the non-naturally encoded amino acid isincorporated into the peptide chain as translated from an mRNAcomprising at least one selector codon. The novel BPFIs of the presentinvention that may be made by chemical synthesis may incorporate atleast one non-naturally encoded amino acid during the synthesis process.The non-naturally encoded amino acid may be placed at any position inthe amino acid chain, and may also be located in any portion of thefinished BPFI, including but not limited to, within the biologicallyactive peptide, linker or fusion partner such as albumin or Fc.

Reference to anionic peptide, HR-C or HR-N polypeptides in thisapplication is intended to use them as an example of a peptide orpolypeptide suitable for use in the present invention. Thus, it isunderstood that the modifications and chemistries described herein withreference to anionic peptide, HR-C or HR-N can be equally applied to anyother BPFIs, including but not limited to, those specifically listedherein.

The incorporation of non-natural amino acids, including syntheticnon-native amino acids, substituted amino acids, or one or more D-aminoacids into the heterologous fusion proteins of the present invention maybe advantageous in a number of different ways. D-amino acid-containingpeptides, etc., exhibit increased stability in vitro or in vivo comparedto L-amino acid-containing counterparts. Thus, the construction ofpeptides, etc., incorporating D-amino acids can be particularly usefulwhen greater intracellular stability is desired or required. Morespecifically, D-peptides, etc., are resistant to endogenous peptidasesand proteases, thereby providing improved bioavailability of themolecule, and prolonged lifetimes in vivo when such properties aredesirable. Additionally, D-peptides, etc., cannot be processedefficiently for major histocompatibility complex class II-restrictedpresentation to T helper cells, and are therefore, less likely to inducehumoral immune responses in the whole organism.

III. General Recombinant Nucleic Acid Methods for Use with the Invention

In numerous embodiments of the present invention, nucleic acids encodinga BPFI of interest will be isolated, cloned and often altered usingrecombinant methods. Such embodiments are used, including but notlimited to, for protein expression or during the generation of variants,derivatives, expression cassettes, or other sequences derived from aBPFI. In some embodiments, the sequences encoding the polypeptides ofthe invention are operably linked to a heterologous promoter. Isolationof anionic peptide, HR-C or HR-N and production of anionic peptide, HR-Cor HR-N in host cells is described in, e.g., U.S. Pat. Nos. [ ], whichis incorporated by reference herein.

A nucleotide sequence encoding a BPFI comprising a non-naturally encodedamino acid may be synthesized on the basis of the amino acid sequence ofthe parent polypeptide and then changing the nucleotide sequence so asto effect introduction (i.e., incorporation or substitution) or removal(i.e., deletion or substitution) of the relevant amino acid residue(s).The nucleotide sequence may be conveniently modified by site-directedmutagenesis in accordance with conventional methods. Alternatively, thenucleotide sequence may be prepared by chemical synthesis, including butnot limited to, by using an oligonucleotide synthesizer, whereinoligonucleotides are designed based on the amino acid sequence of thedesired polypeptide, and preferably selecting those codons that arefavored in the host cell in which the recombinant polypeptide will beproduced. For example, several small oligonucleotides coding forportions of the desired polypeptide may be synthesized and assembled byPCR, ligation or ligation chain reaction. See, e.g., Barany, et al.,Proc. Natl. Acad. Sci. 88: 189-193 (1991); U.S. Pat. No. 6,521,427 whichare incorporated by reference herein.

This invention utilizes routine techniques in the field of recombinantgenetics. Basic texts disclosing the general methods of use in thisinvention include Sambrook et al., Molecular Cloning, A LaboratoryManual (3rd ed. 2001); Kriegler, Gene Transfer and Expression: ALaboratory Manual (1990); and Current Protocols in Molecular Biology(Ausubel et al., eds., 1994)).

General texts which describe molecular biological techniques includeBerger and Kimmel, Guide to Molecular Cloning Techniques, Methods inEnzymology volume 152 Academic Press, Inc., San Diego, Calif. (Berger);Sambrook et al., Molecular Cloning—A Laboratory Manual (2nd Ed.), Vol.1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989(“Sambrook”) and Current Protocols in Molecular Biology, F. M. Ausubelet al., eds., Current Protocols, a joint venture between GreenePublishing Associates, Inc. and John Wiley & Sons, Inc., (supplementedthrough 1999) (“Ausubel”)). These texts describe mutagenesis, the use ofvectors, promoters and many other relevant topics related to, includingbut not limited to, the generation of genes that include selector codonsfor production of proteins that include unnatural amino acids,orthogonal tRNAs, orthogonal synthetases, and pairs thereof. Promotersinclude, but are not limited to, a prokaryotic promoter, a eukaryoticpromoter, a bacterial promoter, a yeast promoter, an insect promoter, amammalian promoter, a unique promoter, and an inducible promoter.

Various types of mutagenesis are used in the invention for a variety ofpurposes, including but not limited to, to produce libraries of tRNAs,to produce libraries of synthetases, to produce selector codons, toinsert selector codons that encode unnatural amino acids in a protein orpolypeptide of interest. They include but are not limited tosite-directed, random point mutagenesis, homologous recombination, DNAshuffling or other recursive mutagenesis methods, chimeric construction,mutagenesis using uracil containing templates, oligonucleotide-directedmutagenesis, phosphorothioate-modified DNA mutagenesis, mutagenesisusing gapped duplex DNA or the like, or any combination thereof.Additional suitable methods include point mismatch repair, mutagenesisusing repair-deficient host strains, restriction-selection andrestriction-purification, deletion mutagenesis, mutagenesis by totalgene synthesis, double-strand break repair, and the like. Mutagenesis,including but not limited to, involving chimeric constructs, are alsoincluded in the present invention. In one embodiment, mutagenesis can beguided by known information of the naturally occurring molecule oraltered or mutated naturally occurring molecule, including but notlimited to, sequence, sequence comparisons, physical properties, crystalstructure or the like.

The texts and examples found herein describe these procedures.Additional information is found in the following publications andreferences cited within: Ling et al., Approaches to DNA mutagenesis: anoverview, Anal Biochem. 254(2): 157-178 (1997); Dale et al.,Oligonucleotide-directed random mutagenesis using the phosphorothioatemethod, Methods Mol. Biol. 57:369-374 (1996); Smith, In vitromutagenesis, Ann. Rev. Genet. 19:423-462 (1985); Botstein & Shortie,Strategies and applications of in vitro mutagenesis, Science229:1193-1201 (1985); Carter, Site-directed mutagenesis, Biochem. J.237:1-7 (1986); Kunkel, The efficiency of oligonucleotide directedmutagenesis, in Nucleic Acids & Molecular Biology (Eckstein, F. andLilley, D. M. J. eds., Springer Verlag, Berlin) (1987); Kunkel, Rapidand efficient site-specific mutagenesis without phenotypic selection,Proc. Natl. Acad. Sci. USA 82:488-492 (1985); Kunkel et al., Rapid andefficient site-specific mutagenesis without phenotypic selection,Methods in Enzymol. 154, 367-382 (1987); Bass et al., Mutant Trprepressors with new DNA-binding specificities, Science 242:240-245(1988); Zoller & Smith, Oligonucleotide-directed mutagenesis usingM13-derived vectors: an efficient and general procedure for theproduction of point mutations in any DNA fragment, Nucleic Acids Res.10:6487-6500 (1982); Zoller & Smith, Oligonucleotide-directedmutagenesis of DNA fragments cloned into M13 vectors, Methods inEnzymol. 100:468-500 (1983); Zoller & Smith, Oligonucleotide-directedmutagenesis: a simple method using two oligonucleotide primers and asingle-stranded DNA template, Methods in Enzymol. 154:329-350 (1987);Taylor et al., The use of phosphorothioate-modified DNA in restrictionenzyme reactions to prepare nicked DNA, Nucl. Acids Res. 13: 8749-8764(1985); Taylor et al., The rapid generation of oligonucleotide-directedmutations at high frequency using phosphorothioate-modified DNA, Nucl.Acids Res. 13: 8765-8785 (1985); Nakamaye & Eckstein, Inhibition ofrestriction endonuclease Nci I cleavage by phosphorothioate groups andits application to oligonucleotide-directed mutagenesis, Nucl. AcidsRes. 14: 9679-9698 (1986); Sayers et al., 5′-3′ Exonucleases inphosphorothioate-based oligonucleotide-directed mutagenesis, Nucl. AcidsRes. 16:791-802 (1988); Sayers et al., Strand specific cleavage ofphosphorothioate-containing DNA by reaction with restrictionendonucleases in the presence of ethidium bromide, (1988) Nucl. AcidsRes. 16: 803-814; Kramer et al., The gapped duplex DNA approach tooligonucleotide-directed mutation construction, Nucl. Acids Res. 12:9441-9456 (1984); Kramer & Fritz Oligonucleotide-directed constructionof mutations via gapped duplex DNA, Methods in Enzymol. 154:350-367(1987); Kramer et al., Improved enzymatic in vitro reactions in thegapped duplex DNA approach to oligonucleotide-directed construction ofmutations, Nucl. Acids Res. 16: 7207 (1988); Fritz et al.,Oligonucleotide-directed construction of mutations: a gapped duplex DNAprocedure without enzymatic reactions in vitro, Nucl. Acids Res. 16:6987-6999 (1988); Kramer et al., Different base/base mismatches arecorrected with different efficiencies by the methyl-directed DNAmismatch-repair system of E. coli, Cell 38:879-887 (1984); Carter etal., Improved oligonucleotide site-directed mutagenesis using M13vectors, Nucl. Acids Res. 13: 4431-4443 (1985); Carter, Improvedoligonucleotide-directed mutagenesis using M13 vectors, Methods inEnzymol. 154: 382-403 (1987); Eghtedarzadeh & Henikoff, Use ofoligonucleotides to generate large deletions, Nucl. Acids Res. 14: 5115(1986); Wells et al., Importance of hydrogen-bond formation instabilizing the transition state of subtilisin, Phil. Trans. R. Soc.Lond. A 317: 415-423 (1986); Nambiar et al., Total synthesis and cloningof a gene coding for the ribonuclease S protein, Science 223: 1299-1301(1984); Sakmar and Khorana, Total synthesis and expression of a gene forthe alpha-subunit of bovine rod outer segment guanine nucleotide-bindingprotein (transducin), Nucl. Acids Res. 14: 6361-6372 (1988); Wells etal., Cassette mutagenesis: an efficient method for generation ofmultiple mutations at defined sites, Gene 34:315-323 (1985); Grundströmet al., Oligonucleotide-directed mutagenesis by microscale ‘shot-gun’gene synthesis, Nucl. Acids Res. 13: 3305-3316 (1985); Mandecki,Oligonucleotide-directed double-strand break repair in plasmids ofEscherichia coli: a method for site-specific mutagenesis, Proc. Natl.Acad. Sci. USA, 83:7177-7181 (1986); Arnold, Protein engineering forunusual environments, Current Opinion in Biotechnology 4:450-455 (1993);Sieber, et al., Nature Biotechnology, 19:456-460 (2001); W. P. C.Stemmer, Nature 370, 389-91 (1994); and, I. A. Lorimer, I. Pastan,Nucleic Acids Res. 23, 3067-8 (1995). Additional details on many of theabove methods can be found in Methods in Enzymology Volume 154, whichalso describes useful controls for trouble-shooting problems withvarious mutagenesis methods.

The invention also relates to eukaryotic host cells, non-eukaryotic hostcells, and organisms for the in vivo incorporation of an unnatural aminoacid via orthogonal tRNA/RS pairs. Host cells are genetically engineered(including but not limited to, transformed, transduced or transfected)with the polynucleotides of the invention or constructs which include apolynucleotide of the invention, including but not limited to, a vectorof the invention, which can be, for example, a cloning vector or anexpression vector. The vector can be, for example, in the form of aplasmid, a bacterium, a virus, a naked polynucleotide, or a conjugatedpolynucleotide. The vectors are introduced into cells and/ormicroorganisms by standard methods including electroporation (Fromm etal., Proc. Natl. Acad. Sci. USA 82, 5824 (1985), infection by viralvectors, high velocity ballistic penetration by small particles with thenucleic acid either within the matrix of small beads or particles, or onthe surface (Klein et al., Nature 327, 70-73 (1987)).

The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for such activities as, for example, screeningsteps, activating promoters or selecting transformants. These cells canoptionally be cultured into transgenic organisms. Other usefulreferences, including but not limited to for cell isolation and culture(e.g., for subsequent nucleic acid isolation) include Freshney (1994)Culture of Animal Cells, a Manual of Basic Technique, third edition,Wiley-Liss, New York and the references cited therein; Payne et al.(1992) Plant Cell and Tissue Culture in Liquid Systems John Wiley &Sons, Inc. New York, N.Y.; Gamborg and Phillips (eds.) (1995) PlantCell, Tissue and Organ Culture; Fundamental Methods Springer Lab Manual,Springer-Verlag (Berlin Heidelberg New York) and Atlas and Parks (eds.)The Handbook of Microbiological Media (1993) CRC Press, Boca Raton, Fla.

Several well-known methods of introducing target nucleic acids intocells are available, any of which can be used in the invention. Theseinclude: fusion of the recipient cells with bacterial protoplastscontaining the DNA, electroporation, projectile bombardment, andinfection with viral vectors (discussed further, below), etc. Bacterialcells can be used to amplify the number of plasmids containing DNAconstructs of this invention. The bacteria are grown to log phase andthe plasmids within the bacteria can be isolated by a variety of methodsknown in the art (see, for instance, Sambrook). In addition, a plethoraof kits are commercially available for the purification of plasmids frombacteria, (see, e.g., EasyPrep™, FlexiPrep™, both from PharmaciaBiotech; StrataClean™ from Stratagene; and, QIAprep™ from Qiagen). Theisolated and purified plasmids are then further manipulated to produceother plasmids, used to transfect cells or incorporated into relatedvectors to infect organisms. Typical vectors contain transcription andtranslation terminators, transcription and translation initiationsequences, and promoters useful for regulation of the expression of theparticular target nucleic acid. The vectors optionally comprise genericexpression cassettes containing at least one independent terminatorsequence, sequences permitting replication of the cassette ineukaryotes, or prokaryotes, or both, (including but not limited to,shuttle vectors) and selection markers for both prokaryotic andeukaryotic systems. Vectors are suitable for replication and integrationin prokaryotes, eukaryotes, or preferably both. See, Gillam & Smith,Gene 8:81 (1979); Roberts, et al., Nature, 328:731 (1987); Schneider,E., et al., Protein Expr. Purif. 6(1)10-14 (1995); Ausubel, Sambrook,Berger (all supra). A catalogue of bacteria and bacteriophages usefulfor cloning is provided, e.g., by the ATCC, e.g., The ATCC Catalogue ofBacteria and Bacteriophage (1992) Gherna et al. (eds) published by theATCC. Additional basic procedures for sequencing, cloning and otheraspects of molecular biology and underlying theoretical considerationsare also found in Watson et al. (1992) Recombinant DNA Second EditionScientific American Books, NY. In addition, essentially any nucleic acid(and virtually any labeled nucleic acid, whether standard ornon-standard) can be custom or standard ordered from any of a variety ofcommercial sources, such as the Midland Certified Reagent Company(Midland, Tex. available on the World Wide Web at mcrc.com), The GreatAmerican Gene Company (Ramona, Calif. available on the World Wide Web atgenco.com), ExpressGen Inc. (Chicago, Ill. available on the World WideWeb at expressgen.com), Operon Technologies Inc. (Alameda, Calif.) andmany others.

Selector Codons

Selector codons of the invention expand the genetic codon framework ofprotein biosynthetic machinery. For example, a selector codon includes,but is not limited to, a unique three base codon, a nonsense codon, suchas a stop codon, including but not limited to, an amber codon (UAG), oran opal codon (UGA), an unnatural codon, a four or more base codon, arare codon, or the like. It is readily apparent to those of ordinaryskill in the art that there is a wide range in the number of selectorcodons that can be introduced into a desired gene, including but notlimited to, one or more, two or more, more than three, 4, 5, 6, 7, 8, 9,10 or more in a single polynucleotide encoding at least a portion of theBPFI.

In one embodiment, the methods involve the use of a selector codon thatis a stop codon for the incorporation of unnatural amino acids in vivoin a eukaryotic cell. For example, an O-tRNA is produced that recognizesthe stop codon, including but not limited to, UAG, and is aminoacylatedby an O-RS with a desired unnatural amino acid. This O-tRNA is notrecognized by the naturally occurring host's aminoacyl-tRNA synthetases.Conventional site-directed mutagenesis can be used to introduce the stopcodon, including but not limited to, TAG, at the site of interest in apolypeptide of interest. See, e.g., Sayers, J. R., et al. (1988), 5′-3′Exonucleases in phosphorothioate-based oligonucleotide-directedmutagenesis. Nucleic Acids Res 16:791-802. When the O-RS, O-tRNA and thenucleic acid that encodes the polypeptide of interest are combined invivo, the unnatural amino acid is incorporated in response to the UAGcodon to give a polypeptide containing the unnatural amino acid at thespecified position.

The incorporation of unnatural amino acids in vivo can be done withoutsignificant perturbation of the eukaryotic host cell. For example,because the suppression efficiency for the UAG codon depends upon thecompetition between the O-tRNA, including but not limited to, the ambersuppressor tRNA, and a eukaryotic release factor (including but notlimited to, eRF) (which binds to a stop codon and initiates release ofthe growing peptide from the ribosome), the suppression efficiency canbe modulated by, including but not limited to, increasing the expressionlevel of O-tRNA, and/or the suppressor tRNA.

Selector codons also comprise extended codons, including but not limitedto, four or more base codons, such as, four, five, six or more basecodons. Examples of four base codons include, including but not limitedto, AGGA, CUAG, UAGA, CCCU and the like. Examples of five base codonsinclude, but are not limited to, AGGAC, CCCCU, CCCUC, CUAGA, CUACU,UAGGC and the like. A feature of the invention includes using extendedcodons based on frameshift suppression. Four or more base codons caninsert, including but not limited to, one or multiple unnatural aminoacids into the same protein. For example, in the presence of mutatedO-tRNAs, including but not limited to, a special frameshift suppressortRNAs, with anticodon loops, for example, with at least 8-10 ntanticodon loops, the four or more base codon is read as single aminoacid. In other embodiments, the anticodon loops can decode, includingbut not limited to, at least a four-base codon, at least a five-basecodon, or at least a six-base codon or more. Since there are 256possible four-base codons, multiple unnatural amino acids can be encodedin the same cell using a four or more base codon. See, Anderson et al.,(2002) Exploring the Limits of Codon and Anticodon Size, Chemistry andBiology, 9:237-244; Magliery, (2001) Expanding the Genetic Code:Selection of Efficient Suppressors of Four-base Codons andIdentification of “Shifty” Four-base Codons with a Library Approach inEscherichia coli, J. Mol. Biol. 307: 755-769.

For example, four-base codons have been used to incorporate unnaturalamino acids into proteins using in vitro biosynthetic methods. See,e.g., Ma et al., (1993) Biochemistry, 32:7939; and Hohsaka et al.,(1999) J. Am. Chem. Soc., 121:34. CGGG and AGGU were used tosimultaneously incorporate 2-naphthylalanine and an NBD derivative oflysine into streptavidin in vitro with two chemically acylatedframeshift suppressor tRNAs. See, e.g., Hohsaka et al., (1999) J. Am.Chem. Soc., 121:12194. In an in vivo study, Moore et al. examined theability of tRNALeu derivatives with NCUA anticodons to suppress UAGNcodons (N can be U, A, G, or C), and found that the quadruplet UAGA canbe decoded by a tRNALeu with a UCUA anticodon with an efficiency of 13to 26% with little decoding in the 0 or −1 frame. See, Moore et al.,(2000) J. Mol. Biol., 298:195. In one embodiment, extended codons basedon rare codons or nonsense codons can be used in the present invention,which can reduce missense readthrough and frameshift suppression atother unwanted sites.

For a given system, a selector codon can also include one of the naturalthree base codons, where the endogenous system does not use (or rarelyuses) the natural base codon. For example, this includes a system thatis lacking a tRNA that recognizes the natural three base codon, and/or asystem where the three base codon is a rare codon.

Selector codons optionally include unnatural base pairs. These unnaturalbase pairs further expand the existing genetic alphabet. One extra basepair increases the number of triplet codons from 64 to 125. Propertiesof third base pairs include stable and selective base pairing, efficientenzymatic incorporation into DNA with high fidelity by a polymerase, andthe efficient continued primer extension after synthesis of the nascentunnatural base pair. Descriptions of unnatural base pairs which can beadapted for methods and compositions include, e.g., Hirao, et al.,(2002) An unnatural base pair for incorporating amino acid analoguesinto protein, Nature Biotechnology, 20:177-182. Other relevantpublications are listed below.

For in vivo usage, the unnatural nucleoside is membrane permeable and isphosphorylated to form the corresponding triphosphate. In addition, theincreased genetic information is stable and not destroyed by cellularenzymes. Previous efforts by Benner and others took advantage ofhydrogen bonding patterns that are different from those in canonicalWatson-Crick pairs, the most noteworthy example of which is theiso-C:iso-G pair. See, e.g., Switzer et al., (1989) J. Am. Chem. Soc.,111:8322; and Piccirilli et al., (1990) Nature, 343:33; Kool, (2000)Curr. Opin. Chem. Biol., 4:602. These bases in general mispair to somedegree with natural bases and cannot be enzymatically replicated. Kooland co-workers demonstrated that hydrophobic packing interactionsbetween bases can replace hydrogen bonding to drive the formation ofbase pair. See, Kool, (2000) Curr. Opin. Chem. Biol., 4:602; and Guckianand Kool, (1998) Angew. Chem. Int. Ed. Engl., 36, 2825. In an effort todevelop an unnatural base pair satisfying all the above requirements,Schultz, Romesberg and co-workers have systematically synthesized andstudied a series of unnatural hydrophobic bases. A PICS:PICS self-pairis found to be more stable than natural base pairs, and can beefficiently incorporated into DNA by Klenow fragment of Escherichia coliDNA polymerase I (KF). See, e.g., McMinn et al., (1999) J. Am. Chem.Soc., 121:11585-6; and Ogawa et al., (2000) J. Am. Chem. Soc., 122:3274.A 3MN:3MN self-pair can be synthesized by KF with efficiency andselectivity sufficient for biological function. See, e.g., Ogawa et al.,(2000) J. Am. Chem. Soc., 122:8803. However, both bases act as a chainterminator for further replication. A mutant DNA polymerase has beenrecently evolved that can be used to replicate the PICS self pair. Inaddition, a 7AI self pair can be replicated. See, e.g., Tae et al.,(2001) J. Am. Chem. Soc., 123:7439. A novel metallobase pair, Dipic:Py,has also been developed, which forms a stable pair upon binding Cu(II).See, Meggers et al., (2000) J. Am. Chem. Soc., 122:10714. Becauseextended codons and unnatural codons are intrinsically orthogonal tonatural codons, the methods of the invention can take advantage of thisproperty to generate orthogonal tRNAs for them.

A translational bypassing system can also be used to incorporate anunnatural amino acid in a desired polypeptide. In a translationalbypassing system, a large sequence is incorporated into a gene but isnot translated into protein. The sequence contains a structure thatserves as a cue to induce the ribosome to hop over the sequence andresume translation downstream of the insertion.

In certain embodiments, the protein or polypeptide of interest (orportion thereof) in the methods and/or compositions of the invention isencoded by a nucleic acid. Typically, the nucleic acid comprises atleast one selector codon, at least two selector codons, at least threeselector codons, at least four selector codons, at least five selectorcodons, at least six selector codons, at least seven selector codons, atleast eight selector codons, at least nine selector codons, ten or moreselector codons.

Genes coding for proteins or polypeptides of interest can be mutagenizedusing methods well-known to one of skill in the art and described hereinto include, for example, one or more selector codon for theincorporation of an unnatural amino acid. For example, a nucleic acidfor a protein of interest is mutagenized to include one or more selectorcodon, providing for the incorporation of one or more unnatural aminoacids. The invention includes any such variant, including but notlimited to, mutant, versions of any protein, for example, including atleast one unnatural amino acid. Similarly, the invention also includescorresponding nucleic acids, i.e., any nucleic acid with one or moreselector codon that encodes one or more unnatural amino acid.

Nucleic acid molecules encoding a BPFI such as anionic peptide, HR-C orHR-N may be readily mutated to introduce a cysteine at any desiredposition of the polypeptide. Cysteine is widely used to introducereactive molecules, water soluble polymers, proteins, or a wide varietyof other molecules, onto a protein of interest. Methods suitable for theincorporation of cysteine into a desired position of a polypeptide arewell known in the art, such as those described in U.S. Pat. No.6,608,183, and include standard mutagenesis techniques.

IV. Non-Naturally Encoded Amino Acids

A very wide variety of non-naturally encoded amino acids are suitablefor use in the present invention. Any number of non-naturally encodedamino acids can be introduced into a BPFI. In general, the introducednon-naturally encoded amino acids are substantially chemically inerttoward the 20 common, genetically-encoded amino acids (i.e., alanine,arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid,glycine, histidine, isoleucine, leucine, lysine, methionine,phenylalanine, proline, serine, threonine, tryptophan, tyrosine, andvaline). In some embodiments, the non-naturally encoded amino acidsinclude side chain functional groups that react efficiently andselectively with functional groups not found in the 20 common aminoacids (including but not limited to, azido, ketone, aldehyde andaminooxy groups) to form stable conjugates. For example, a BPFI thatincludes a non-naturally encoded amino acid containing an azidofunctional group can be reacted with a polymer (including but notlimited to, poly(ethylene glycol) or, alternatively, a secondpolypeptide containing an alkyne moiety to form a stable conjugateresulting for the selective reaction of the azide and the alkynefunctional groups to form a Huisgen [3+2] cycloaddition product.

The generic structure of an alpha-amino acid is illustrated as follows(Formula I):

A non-naturally encoded amino acid is typically any structure having theabove-listed formula wherein the R group is any substituent other thanone used in the twenty natural amino acids, and may be suitable for usein the present invention. Because the non-naturally encoded amino acidsof the invention typically differ from the natural amino acids only inthe structure of the side chain, the non-naturally encoded amino acidsform amide bonds with other amino acids, including but not limited to,natural or non-naturally encoded, in the same manner in which they areformed in naturally occurring polypeptides. However, the non-naturallyencoded amino acids have side chain groups that distinguish them fromthe natural amino acids. For example, R optionally comprises an alkyl-,aryl-, acyl-, keto-, azido-, hydroxyl-, hydrazine, cyano-, halo-,hydrazide, alkenyl, alkynl, ether, thiol, seleno-, sulfonyl-, borate,boronate, phospho, phosphono, phosphine, heterocyclic, enone, imine,aldehyde, ester, thioacid, hydroxylamine, amino group, or the like orany combination thereof. Other non-naturally occurring amino acids ofinterest that may be suitable for use in the present invention include,but are not limited to, amino acids comprising a photoactivatablecross-linker, spin-labeled amino acids, fluorescent amino acids, metalbinding amino acids, metal-containing amino acids, radioactive aminoacids, amino acids with novel functional groups, amino acids thatcovalently or noncovalently interact with other molecules, photocagedand/or photoisomerizable amino acids, amino acids comprising biotin or abiotin analogue, glycosylated amino acids such as a sugar substitutedserine, other carbohydrate modified amino acids, keto-containing aminoacids, amino acids comprising polyethylene glycol or polyether, heavyatom substituted amino acids, chemically cleavable and/or photocleavableamino acids, amino acids with an elongated side chains as compared tonatural amino acids, including but not limited to, polyethers or longchain hydrocarbons, including but not limited to, greater than about 5or greater than about 10 carbons, carbon-linked sugar-containing aminoacids, redox-active amino acids, amino thioacid containing amino acids,and amino acids comprising one or more toxic moiety.

Exemplary non-naturally encoded amino acids that may be suitable for usein the present invention and that are useful for reactions with watersoluble polymers include, but are not limited to, those with carbonyl,aminooxy, hydrazine, hydrazide, semicarbazide, azide and alkyne reactivegroups. In some embodiments, non-naturally encoded amino acids comprisea saccharide moiety. Examples of such amino acids includeN-acetyl-L-glucosaminyl-L-serine, N-acetyl-L-galactosaminyl-L-serine,N-acetyl-L-glucosaminyl-L-threonine,N-acetyl-L-glucosaminyl-L-asparagine and O-mannosaminyl-L-serine.Examples of such amino acids also include examples where thenaturally-occurring N- or O-linkage between the amino acid and thesaccharide is replaced by a covalent linkage not commonly found innature—including but not limited to, an alkene, an oxime, a thioether,an amide and the like. Examples of such amino acids also includesaccharides that are not commonly found in naturally-occurring proteinssuch as 2-deoxy-glucose, 2-deoxygalactose and the like.

Many of the non-naturally encoded amino acids provided herein arecommercially available, e.g., from Sigma-Aldrich (St. Louis, Mo., USA),Novabiochem (a division of EMD Biosciences, Darmstadt, Germany), orPeptech (Burlington, Mass., USA). Those that are not commerciallyavailable are optionally synthesized as provided herein or usingstandard methods known to those of skill in the art. For organicsynthesis techniques, see, e.g., Organic Chemistry by Fessendon andFessendon, (1982, Second Edition, Willard Grant Press, Boston Mass.);Advanced Organic Chemistry by March (Third Edition, 1985, Wiley andSons, New York); and Advanced Organic Chemistry by Carey and Sundberg(Third Edition, Parts A and B, 1990, Plenum Press, New York). See, also,U.S. Patent Application Publications 2003/0082575 and 2003/0108885,which is incorporated by reference herein. In addition to unnaturalamino acids that contain novel side chains, unnatural amino acids thatmay be suitable for use in the present invention also optionallycomprise modified backbone structures, including but not limited to, asillustrated by the structures of Formula II and III:

wherein Z typically comprises OH, NH₂, SH, NH—R′, or S—R′; X and Y,which can be the same or different, typically comprise S or O, and R andR′, which are optionally the same or different, are typically selectedfrom the same list of constituents for the R group described above forthe unnatural amino acids having Formula I as well as hydrogen. Forexample, unnatural amino acids of the invention optionally comprisesubstitutions in the amino or carboxyl group as illustrated by FormulasII and III. Unnatural amino acids of this type include, but are notlimited to, α-hydroxy acids, α-thioacids, α-aminothiocarboxylates,including but not limited to, with side chains corresponding to thecommon twenty natural amino acids or unnatural side chains. In addition,substitutions at the α-carbon optionally include, but are not limitedto, L, D, or α-α-disubstituted amino acids such as D-glutamate,D-alanine, D-methyl-O-tyrosine, aminobutyric acid, and the like. Otherstructural alternatives include cyclic amino acids, such as prolineanalogues as well as 3, 4,6, 7, 8, and 9 membered ring prolineanalogues, β and γ amino acids such as substituted β-alanine and γ-aminobutyric acid.

1209] Many unnatural amino acids are based on natural amino acids, suchas tyrosine, glutamine, phenylalanine, and the like, and are suitablefor use in the present invention. Tyrosine analogs include, but are notlimited to, para-substituted tyrosines, ortho-substituted tyrosines, andmeta substituted tyrosines, where the substituted tyrosine comprises,including but not limited to, a keto group (including but not limitedto, an acetyl group), a benzoyl group, an amino group, a hydrazine, anhydroxyamine, a thiol group, a carboxy group, an isopropyl group, amethyl group, a C₆-C₂₀ straight chain or branched hydrocarbon, asaturated or unsaturated hydrocarbon, an O-methyl group, a polyethergroup, a nitro group, an alkynyl group or the like. In addition,multiply substituted aryl rings are also contemplated. Glutamine analogsthat may be suitable for use in the present invention include, but arenot limited to, α-hydroxy derivatives, γ-substituted derivatives, cyclicderivatives, and amide substituted glutamine derivatives. Examplephenylalanine analogs that may be suitable for use in the presentinvention include, but are not limited to, para-substitutedphenylalanines, ortho-substituted phenyalanines, and meta-substitutedphenylalanines, where the substituent comprises, including but notlimited to, a hydroxy group, a methoxy group, a methyl group, an allylgroup, an aldehyde, an azido, an iodo, a bromo, a keto group (includingbut not limited to, an acetyl group), a benzoyl, an alkynyl group, orthe like. Specific examples of unnatural amino acids that may besuitable for use in the present invention include, but are not limitedto, a p-acetyl-L-phenylalanine, an O-methyl-L-tyrosine, anL-3-(2-naphthyl)alanine, a 3-methyl-phenylalanine, anO-4-allyl-L-tyrosine, a 4-propyl-L-tyrosine, atri-O-acetyl-GlcNAcβ-serine, an L-Dopa, a fluorinated phenylalanine, anisopropyl-L-phenylalanine, a p-azido-L-phenylalanine, ap-acyl-L-phenylalanine, a p-benzoyl-L-phenylalanine, an L-phosphoserine,a phosphonoserine, a phosphonotyrosine, a p-iodo-phenylalanine, ap-bromophenylalanine, a p-amino-L-phenylalanine, anisopropyl-L-phenylalanine, and a p-propargyloxy-phenylalanine, and thelike. Examples of structures of a variety of unnatural amino acids thatmay be suitable for use in the present invention are provided in, forexample, WO 2002/085923 entitled “In vivo incorporation of unnaturalamino acids.” See also Kiick et al., (2002) Incorporation of azides intorecombinant proteins for chemoselective modification by the Staudingerligation, PNAS 99:19-24, for additional methionine analogs.

In one embodiment, compositions of BPFI that include an unnatural aminoacid (such as p-(propargyloxy)-phenyalanine) are provided. Variouscompositions comprising p-(propargyloxy)-phenyalanine and, including butnot limited to, proteins and/or cells, are also provided. In one aspect,a composition that includes the p-(propargyloxy)-phenyalanine unnaturalamino acid, further includes an orthogonal tRNA. The unnatural aminoacid can be bonded (including but not limited to, covalently) to theorthogonal tRNA, including but not limited to, covalently bonded to theorthogonal tRNA though an amino-acyl bond, covalently bonded to a 3′OHor a 2′OH of a terminal ribose sugar of the orthogonal tRNA, etc.

The chemical moieties via unnatural amino acids that can be incorporatedinto proteins offer a variety of advantages and manipulations of theprotein. For example, the unique reactivity of a keto functional groupallows selective modification of proteins with any of a number ofhydrazine- or hydroxylamine-containing reagents in vitro and in vivo. Aheavy atom unnatural amino acid, for example, can be useful for phasingX-ray structure data. The site-specific introduction of heavy atomsusing unnatural amino acids also provides selectivity and flexibility inchoosing positions for heavy atoms. Photoreactive unnatural amino acids(including but not limited to, amino acids with benzophenone andarylazides (including but not limited to, phenylazide) side chains), forexample, allow for efficient in vivo and in vitro photocrosslinking ofprotein. Examples of photoreactive unnatural amino acids include, butare not limited to, p-azido-phenylalanine and p-benzoyl-phenylalanine.The protein with the photoreactive unnatural amino acids can then becrosslinked at will by excitation of the photoreactive group-providingtemporal control. In one example, the methyl group of an unnatural aminocan be substituted with an isotopically labeled, including but notlimited to, methyl group, as a probe of local structure and dynamics,including but not limited to, with the use of nuclear magnetic resonanceand vibrational spectroscopy. Alkynyl or azido functional groups, forexample, allow the selective modification of proteins with moleculesthrough a [3+2] cycloaddition reaction.

A non-natural amino acid incorporated into a polypeptide at the aminoterminus can be composed of an R group that is any substituent otherthan one used in the twenty natural amino acids and a 2^(nd) reactivegroup different from the NH₂ group normally present in α-amino acids(see Formula I). A similar non-natural amino acid can be incorporated atthe carboxyl terminus with a 2^(nd) reactive group different from theCOOH group normally present in α-amino acids (see Formula I).

Chemical Synthesis of Unnatural Amino Acids

Many of the unnatural amino acids suitable for use in the presentinvention are commercially available, e.g., from Sigma (USA) or Aldrich(Milwaukee, Wis., USA). Those that are not commercially available areoptionally synthesized as provided herein or as provided in variouspublications or using standard methods known to those of skill in theart. For organic synthesis techniques, see, e.g., Organic Chemistry byFessendon and Fessendon, (1982, Second Edition, Willard Grant Press,Boston Mass.); Advanced Organic Chemistry by March (Third Edition, 1985,Wiley and Sons, New York); and Advanced Organic Chemistry by Carey andSundberg (Third Edition, Parts A and B, 1990, Plenum Press, New York).Additional publications describing the synthesis of unnatural aminoacids include, e.g., WO 2002/085923 entitled “In vivo incorporation ofUnnatural Amino Acids;” Matsoukas et al., (1995) J. Med. Chem. 38,4660-4669; King, F. E. & Kidd, D. A. A. (1949) A New Synthesis ofGlutamine and of γ-Dipeptides of Glutamic Acid from PhthylatedIntermediates. J. Chem. Soc., 3315-3319; Friedman, O. M. & Chatterrji,R. (1959) Synthesis of Derivatives of Glutamine as Model Substrates forAnti-Tumor Agents. J. Am. Chem. Soc. 81, 3750-3752; Craig, J. C. et al.(1988) Absolute Configuration of the Enantiomers of7-Chloro-[[4-(diethylamino)-1-methylbutyl]amino]quinoline (Chloroquine).J. Org. Chem. 53, 1167-1170; Azoulay, M., Vilmont, M. & Frappier, F.(1991) Glutamine analogues as Potential Antimalarials, Eur. J. Med.Chem. 26, 201-5; Koskinen, A. M. P. & Rapoport, H. (1989) Synthesis of4-Substituted Prolines as Conformationally Constrained Amino AcidAnalogues. J. Org. Chem. 54, 1859-1866; Christie, B. D. & Rapoport, H.(1985) Synthesis of Optically Pure Pipecolates from L-Asparagine.Application to the Total Synthesis of (+)—Apovincamine through AminoAcid Decarbonylation and Iminium Ion Cyclization. J. Org. Chem.50:1239-1246; Barton et al., (1987) Synthesis of Novel alpha-Amino-Acidsand Derivatives Using Radical Chemistry: Synthesis of L- andD-alpha-Amino-Adipic Acids, L-alpha-aminopimelic Acid and AppropriateUnsaturated Derivatives. Tetrahedron 43:4297-4308; and, Subasinghe etal., (1992) Quisqualic acid analogues: synthesis of beta-heterocyclic2-aminopropanoic acid derivatives and their activity at a novelquisqualate-sensitized site. J. Med. Chem. 35:4602-7. See also, patentapplications entitled “Protein Arrays,” filed Dec. 22, 2003, Ser. No.10/744,899 and Ser. No. 60/435,821 filed on Dec. 22, 2002.

A. Carbonyl Reactive Groups

Amino acids with a carbonyl reactive group allow for a variety ofreactions to link molecules (including but not limited to, PEG or otherwater soluble molecules) via nucleophilic addition or aldol condensationreactions among others.

Exemplary carbonyl-containing amino acids can be represented as follows:

wherein n is 0-10; R₁ is an alkyl, aryl, substituted alkyl, orsubstituted aryl; R₂ is H, alkyl, aryl, substituted alkyl, andsubstituted aryl; and R₃ is H, an amino acid, a polypeptide, or an aminoterminus modification group, and R₄ is H, an amino acid, a polypeptide,or a carboxy terminus modification group. In some embodiments, n is 1,R₁ is phenyl and R₂ is a simple alkyl (i.e., methyl, ethyl, or propyl)and the ketone moiety is positioned in the para position relative to thealkyl side chain. In some embodiments, n is 1, R₁ is phenyl and R₂ is asimple alkyl (i.e., methyl, ethyl, or propyl) and the ketone moiety ispositioned in the meta position relative to the alkyl side chain.

The synthesis of p-acetyl-(+/−)-phenylalanine andm-acetyl-(+/−)-phenylalanine is described in Zhang, Z., et al.,Biochemistry 42: 6735-6746 (2003), which is incorporated by referenceherein. Other carbonyl-containing amino acids can be similarly preparedby one skilled in the art.

In some embodiments, a polypeptide comprising a non-naturally encodedamino acid is chemically modified to generate a reactive carbonylfunctional group. For instance, an aldehyde functionality useful forconjugation reactions can be generated from a functionality havingadjacent amino and hydroxyl groups. Where the biologically activemolecule is a polypeptide, for example, an N-terminal serine orthreonine (which may be normally present or may be exposed via chemicalor enzymatic digestion) can be used to generate an aldehydefunctionality under mild oxidative cleavage conditions using periodate.See, e.g., Gaertner, et al., Bioconjug. Chem. 3: 262-268 (1992);Geoghegan, K. & Stroh, J., Bioconjug. Chem. 3:138-146 (1992); Gaertneret al., J. Biol. Chem. 269:7224-7230 (1994). However, methods known inthe art are restricted to the amino acid at the N-terminus of thepeptide or protein.

In the present invention, a non-naturally encoded amino acid bearingadjacent hydroxyl and amino groups can be incorporated into thepolypeptide as a “masked” aldehyde functionality. For example,5-hydroxylysine bears a hydroxyl group adjacent to the epsilon amine.Reaction conditions for generating the aldehyde typically involveaddition of molar excess of sodium metaperiodate under mild conditionsto avoid oxidation at other sites within the polypeptide. The pH of theoxidation reaction is typically about 7.0. A typical reaction involvesthe addition of about 1.5 molar excess of sodium meta periodate to abuffered solution of the polypeptide, followed by incubation for about10 minutes in the dark. See, e.g. U.S. Pat. No. 6,423,685, which isincorporated by reference herein.

The carbonyl functionality can be reacted selectively with a hydrazine-,hydrazide-, hydroxylamine-, or semicarbazide-containing reagent undermild conditions in aqueous solution to form the corresponding hydrazone,oxime, or semicarbazone linkages, respectively, that are stable underphysiological conditions. See, e.g., Jencks, W. P., J. Am. Chem. Soc.81, 475-481 (1959); Shao, J. and Tam, J. P., J. Am. Chem. Soc.117:3893-3899 (1995). Moreover, the unique reactivity of the carbonylgroup allows for selective modification in the presence of the otheramino acid side chains. See, e.g., Cornish, V. W., et al., J. Am. Chem.Soc. 118:8150-8151 (1996); Geoghegan, K. F. & Stroh, J. G., Bioconjug.Chem. 3:138-146 (1992); Mahal, L. K., et al, Science 276:1125-1128(1997).

B. Hydrazine, Hydrazide or Semicarbazide Reactive Groups

Non-naturally encoded amino acids containing a nucleophilic group, suchas a hydrazine, hydrazide or semicarbazide, allow for reaction with avariety of electrophilic groups to form conjugates (including but notlimited to, with PEG or other water soluble polymers).

Exemplary hydrazine, hydrazide or semicarbazide-containing amino acidscan be represented as follows:

wherein n is 0-10; R₁ is an alkyl, aryl, substituted alkyl, orsubstituted aryl or not present; X, is O, N, or S or not present; R₂ isH, an amino acid, a polypeptide, or an amino terminus modificationgroup, and R₃ is H, an amino acid, a polypeptide, or a carboxy terminusmodification group.

In some embodiments, n is 4, R₁ is not present, and X is N. In someembodiments, n is 2, R₁ is not present, and X is not present. In someembodiments, n is 1, R₁ is phenyl, X is O, and the oxygen atom ispositioned para to the alphatic group on the aryl ring.

Hydrazide-, hydrazine-, and semicarbazide-containing amino acids areavailable from commercial sources. For instance, L-glutamate-γ-hydrazideis available from Sigma Chemical (St. Louis, Mo.). Other amino acids notavailable commercially can be prepared by one skilled in the art. See,e.g., U.S. Pat. No. 6,281,211, which is incorporated by referenceherein.

Polypeptides containing non-naturally encoded amino acids that bearhydrazide, hydrazine or semicarbazide functionalities can be reactedefficiently and selectively with a variety of molecules that containaldehydes or other functional groups with similar chemical reactivity.See, e.g., Shao, J. and Tam, J., J. Am. Chem. Soc. 117:3893-3899 (1995).The unique reactivity of hydrazide, hydrazine and semicarbazidefunctional groups makes them significantly more reactive towardaldehydes, ketones and other electrophilic groups as compared to thenucleophilic groups present on the 20 common amino acids (including butnot limited to, the hydroxyl group of serine or threonine or the aminogroups of lysine and the N-terminus).

C. Aminooxy-Containing Amino Acids

Non-naturally encoded amino acids containing an aminooxy (also called ahydroxylamine) group allow for reaction with a variety of electrophilicgroups to form conjugates (including but not limited to, with PEG orother water soluble polymers). Like hydrazines, hydrazides andsemicarbazides, the enhanced nucleophilicity of the aminooxy grouppermits it to react efficiently and selectively with a variety ofmolecules that contain aldehydes or other functional groups with similarchemical reactivity. See, e.g., Shao, J. and Tam, J., J. Am. Chem. Soc.117:3893-3899 (1995); H. Hang and C. Bertozzi, Acc. Chem. Res. 34:727-736 (2001). Whereas the result of reaction with a hydrazine group isthe corresponding hydrazone, however, an oxime results generally fromthe reaction of an aminooxy group with a carbonyl-containing group suchas a ketone.

Exemplary amino acids containing aminooxy groups can be represented asfollows:

wherein n is 0-10; R₁ is an alkyl, aryl, substituted alkyl, orsubstituted aryl or not present; X is O, N, S or not present; m is 0-10;Y═C(O) or not present; R₂ is H, an amino acid, a polypeptide, or anamino terminus modification group, and R₃ is H, an amino acid, apolypeptide, or a carboxy terminus modification group. In someembodiments, n is 1, R₁ is phenyl, X is O, m is 1, and Y is present. Insome embodiments, n is 2, R₁ and X are not present, m is 0, and Y is notpresent.

Aminooxy-containing amino acids can be prepared from readily availableamino acid precursors (homoserine, serine and threonine). See, e.g., M.Carrasco and R. Brown, J. Org. Chem. 68: 8853-8858 (2003). Certainaminooxy-containing amino acids, such as L-2-amino-4-(aminooxy)butyricacid), have been isolated from natural sources (Rosenthal, G., Life Sci.60: 1635-1641 (1997). Other aminooxy-containing amino acids can beprepared by one skilled in the art.

D. Azide and Alkyne Reactive Groups

The unique reactivity of azide and alkyne functional groups makes themextremely useful for the selective modification of polypeptides andother biological molecules. Organic azides, particularly alphaticazides, and alkynes are generally stable toward common reactive chemicalconditions. In particular, both the azide and the alkyne functionalgroups are inert toward the side chains (i.e., R groups) of the 20common amino acids found in naturally-occurring polypeptides. Whenbrought into close proximity, however, the “spring-loaded” nature of theazide and alkyne groups is revealed and they react selectively andefficiently via Huisgen [3+2] cycloaddition reaction to generate thecorresponding triazole. See, e.g., Chin J., et al., Science 301:964-7(2003); Wang, Q., et al., J. Am. Chem. Soc. 125, 3192-3193 (2003); Chin,J. W., et al., J. Am. Chem. Soc. 124:9026-9027 (2002).

Because the Huisgen cycloaddition reaction involves a selectivecycloaddition reaction (see, e.g., Padwa, A., in COMPREHENSIVE ORGANICSYNTHESIS, Vol. 4, (ed. Trost, B. M., 1991), p. 1069-1109; Huisgen, R.in 1,3-DIPOLAR CYCLOADDITION CHEMISTRY, (ed. Padwa, A., 1984), p. 1-176)rather than a nucleophilic substitution, the incorporation ofnon-naturally encoded amino acids bearing azide and alkyne-containingside chains permits the resultant polypeptides to be modifiedselectively at the position of the non-naturally encoded amino acid.Cycloaddition reaction involving azide or alkyne-containing BSP can becarried out at room temperature under aqueous conditions by the additionof Cu(II) (including but not limited to, in the form of a catalyticamount of CuSO₄) in the presence of a reducing agent for reducing Cu(II)to Cu(I), in situ, in catalytic amount. See, e.g., Wang, Q., et al., J.Am. Chem. Soc. 125, 3192-3193 (2003); Tornoe, C. W., et al., J. Org.Chem. 67:3057-3064 (2002); Rostovtsev, et al., Angew. Chem. Int. Ed.41:2596-2599 (2002). Exemplary reducing agents include, including butnot limited to, ascorbate, metallic copper, quinine, hydroquinone,vitamin K, glutathione, cysteine, Fe²⁺, Co²⁺, and an applied electricpotential.

In some cases, where a Huisgen [3+2] cycloaddition reaction between anazide and an alkyne is desired, the BSP comprises a non-naturallyencoded amino acid comprising an alkyne moiety and the water solublepolymer to be attached to the amino acid comprises an azide moiety.Alternatively, the converse reaction (i.e., with the azide moiety on theamino acid and the alkyne moiety present on the water soluble polymer)can also be performed.

The azide functional group can also be reacted selectively with a watersoluble polymer containing an aryl ester and appropriatelyfunctionalized with an aryl phosphine moiety to generate an amidelinkage. The aryl phosphine group reduces the azide in situ and theresulting amine then reacts efficiently with a proximal ester linkage togenerate the corresponding amide. See, e.g., E. Saxon and C. Bertozzi,Science 287, 2007-2010 (2000). The azide-containing amino acid can beeither an alkyl azide (including but not limited to,2-amino-6-azido-1-hexanoic acid) or an aryl azide(p-azido-phenylalanine).

Exemplary water soluble polymers containing an aryl ester and aphosphine moiety can be represented as follows:

wherein X can be O, N, S or not present, Ph is phenyl, W is a watersoluble polymer and R can be H, alkyl, aryl, substituted alkyl andsubstituted aryl groups. Exemplary R groups include but are not limitedto —CH₂, —C(CH₃)₃, —OR′, —NR′R″, —SR′, -halogen, —C(O)R′, —CONR′R″,—S(O)₂R′, —S(O)₂NR′R″, —CN and —NO₂. R′, R″, R′″ and R″″ eachindependently refer to hydrogen, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, including but notlimited to, aryl substituted with 1-3 halogens, substituted orunsubstituted alkyl, alkoxy or thioalkoxy groups, or arylalkyl groups.When a compound of the invention includes more than one R group, forexample, each of the R groups is independently selected as are each R′,R″, R′″ and R″″ groups when more than one of these groups is present.When R′ and R″ are attached to the same nitrogen atom, they can becombined with the nitrogen atom to form a 5-, 6-, or 7-membered ring.For example, —NR′R″ is meant to include, but not be limited to,1-pyrrolidinyl and 4-morpholinyl. From the above discussion ofsubstituents, one of skill in the art will understand that the term“alkyl” is meant to include groups including carbon atoms bound togroups other than hydrogen groups, such as haloalkyl (including but notlimited to, —CF₃ and —CH₂CF₃) and acyl (including but not limited to,—C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and the like).

The azide functional group can also be reacted selectively with a watersoluble polymer containing a thioester and appropriately functionalizedwith an aryl phosphine moiety to generate an amide linkage. The arylphosphine group reduces the azide in situ and the resulting amine thenreacts efficiently with the thioester linkage to generate thecorresponding amide. Exemplary water soluble polymers containing athioester and a phosphine moiety can be represented as follows:

wherein n is 1-10; X can be O, N, S or not present, Ph is phenyl, and Wis a water soluble polymer.

Exemplary alkyne-containing amino acids can be represented as follows:

wherein n is 0-10; R₁ is an alkyl, aryl, substituted alkyl, orsubstituted aryl or not present; X is O, N, S or not present; m is 0-10,R₂ is H, an amino acid, a polypeptide, or an amino terminus modificationgroup, and R₃ is H, an amino acid, a polypeptide, or a carboxy terminusmodification group. In some embodiments, n is 1, R₁ is phenyl, X is notpresent, m is 0 and the acetylene moiety is positioned in the paraposition relative to the alkyl side chain. In some embodiments, n is 1,R₁ is phenyl, X is O, m is 1 and the propargyloxy group is positioned inthe para position relative to the alkyl side chain (i.e.,O-propargyl-tyrosine). In some embodiments, n is 1, R₁ and X are notpresent and m is 0 (i.e., proparylglycine).

Alkyne-containing amino acids are commercially available. For example,propargylglycine is commercially available from Peptech (Burlington,Mass.). Alternatively, alkyne-containing amino acids can be preparedaccording to standard methods. For instance, p-propargyloxyphenylalaninecan be synthesized, for example, as described in Deiters, A., et al., J.Am. Chem. Soc. 125: 11782-11783 (2003), and 4-alkynyl-L-phenylalaninecan be synthesized as described in Kayser, B., et al., Tetrahedron53(7): 2475-2484 (1997). Other alkyne-containing amino acids can beprepared by one skilled in the art.

Exemplary azide-containing amino acids can be represented as follows:

wherein n is 0-10; R₁ is an alkyl, aryl, substituted alkyl, substitutedaryl or not present; X is O, N, S or not present; m is 0-10; R₂ is H, anamino acid, a polypeptide, or an amino terminus modification group, andR₃ is H, an amino acid, a polypeptide, or a carboxy terminusmodification group. In some embodiments, n is 1, R₁ is phenyl, X is notpresent, m is 0 and the azide moiety is positioned para to the alkylside chain. In some embodiments, n is 0-4 and R₁ and X are not present,and m=0. In some embodiments, n is 1, R₁ is phenyl, X is O, m is 2 andthe β-azidoethoxy moiety is positioned in the para position relative tothe alkyl side chain.

Azide-containing amino acids are available from commercial sources. Forinstance, 4-azidophenylalanine can be obtained from Chem-ImpexInternational, Inc. (Wood Dale, Ill.). For those azide-containing aminoacids that are not commercially available, the azide group can beprepared relatively readily using standard methods known to those ofskill in the art, including but not limited to, via displacement of asuitable leaving group (including but not limited to, halide, mesylate,tosylate) or via opening of a suitably protected lactone. See, e.g.,Advanced Organic Chemistry by March (Third Edition, 1985, Wiley andSons, New York).

E. Aminothiol Reactive Groups

The unique reactivity of beta-substituted aminothiol functional groupsmakes them extremely useful for the selective modification ofpolypeptides and other biological molecules that contain aldehyde groupsvia formation of the thiazolidine. See, e.g., J. Shao and J. Tam, J. Am.Chem. Soc. 1995, 117 (14) 3893-3899. In some embodiments,beta-substituted aminothiol amino acids can be incorporated into BSPsand then reacted with water soluble polymers comprising an aldehydefunctionality. In some embodiments, a water soluble polymer, drugconjugate or other payload can be coupled to a BSP comprising abeta-substituted aminothiol amino acid via formation of thethiazolidine.

Cellular Uptake of Unnatural Amino Acids

Unnatural amino acid uptake by a eukaryotic cell is one issue that istypically considered when designing and selecting unnatural amino acids,including but not limited to, for incorporation into a protein. Forexample, the high charge density of α-amino acids suggests that thesecompounds are unlikely to be cell permeable. Natural amino acids aretaken up into the eukaryotic cell via a collection of protein-basedtransport systems. A rapid screen can be done which assesses whichunnatural amino acids, if any, are taken up by cells. See, e.g., thetoxicity assays in, e.g., the applications entitled “Protein Arrays,”filed Dec. 22, 2003, Ser. No. 10/744,899 and Ser. No. 60/435,821 filedon Dec. 22, 2002; and Liu, D. R. & Schultz, P. G. (1999) Progress towardthe evolution of an organism with an expanded genetic code. PNAS UnitedStates 96:4780-4785. Although uptake is easily analyzed with variousassays, an alternative to designing unnatural amino acids that areamenable to cellular uptake pathways is to provide biosynthetic pathwaysto create amino acids in vivo.

Biosynthesis of Unnatural Amino Acids

Many biosynthetic pathways already exist in cells for the production ofamino acids and other compounds. While a biosynthetic method for aparticular unnatural amino acid may not exist in nature, including butnot limited to, in a eukaryotic cell, the invention provides suchmethods. For example, biosynthetic pathways for unnatural amino acidsare optionally generated in host cell by adding new enzymes or modifyingexisting host cell pathways. Additional new enzymes are optionallynaturally occurring enzymes or artificially evolved enzymes. Forexample, the biosynthesis of p-aminophenylalanine (as presented in anexample in WO 2002/085923 entitled “In vivo incorporation of unnaturalamino acids”) relies on the addition of a combination of known enzymesfrom other organisms. The genes for these enzymes can be introduced intoa eukaryotic cell by transforming the cell with a plasmid comprising thegenes. The genes, when expressed in the cell, provide an enzymaticpathway to synthesize the desired compound. Examples of the types ofenzymes that are optionally added are provided in the examples below.Additional enzymes sequences are found, for example, in Genbank.Artificially evolved enzymes are also optionally added into a cell inthe same manner. In this manner, the cellular machinery and resources ofa cell are manipulated to produce unnatural amino acids.

A variety of methods are available for producing novel enzymes for usein biosynthetic pathways or for evolution of existing pathways. Forexample, recursive recombination, including but not limited to, asdeveloped by Maxygen, Inc. (available on the World Wide Web atmaxygen.com), is optionally used to develop novel enzymes and pathways.See, e.g., Stemmer (1994), Rapid evolution of a protein in vitro by DNAshuffling, Nature 370(4):389-391; and, Stemmer, (1994), DNA shuffling byrandom fragmentation and reassembly: In vitro recombination formolecular evolution, Proc. Natl. Acad. Sci. USA., 91:10747-10751.Similarly DesignPath™, developed by Genencor (available on the WorldWide Web at genencor.com) is optionally used for metabolic pathwayengineering, including but not limited to, to engineer a pathway tocreate O-methyl-L-tyrosine in a cell. This technology reconstructsexisting pathways in host organisms using a combination of new genes,including but not limited to, identified through functional genomics,and molecular evolution and design. Diversa Corporation (available onthe World Wide Web at diversa.com) also provides technology for rapidlyscreening libraries of genes and gene pathways, including but notlimited to, to create new pathways.

Typically, the unnatural amino acid produced with an engineeredbiosynthetic pathway of the invention is produced in a concentrationsufficient for efficient protein biosynthesis, including but not limitedto, a natural cellular amount, but not to such a degree as to affect theconcentration of the other amino acids or exhaust cellular resources.Typical concentrations produced in vivo in this manner are about 10 mMto about 0.05 mM. Once a cell is transformed with a plasmid comprisingthe genes used to produce enzymes desired for a specific pathway and anunnatural amino acid is generated, in vivo selections are optionallyused to further optimize the production of the unnatural amino acid forboth ribosomal protein synthesis and cell growth.

Polypeptides with Unnatural Amino Acids

The incorporation of an unnatural amino acid can be done for a varietyof purposes, including but not limited to, tailoring changes in proteinstructure and/or function, changing size, acidity, nucleophilicity,hydrogen bonding, hydrophobicity, accessibility of protease targetsites, targeting to a moiety (including but not limited to, for aprotein array), adding a biologically active molecule, attaching apolymer, attaching a radionuclide, modulating serum half-life,modulating tissue penetration (e.g. tumors), modulating activetransport, modulating tissue, cell or organ specificity or distribution,modulating immunogenicity, modulating protease resistance, etc. Proteinsthat include an unnatural amino acid can have enhanced or even entirelynew catalytic or biophysical properties. For example, the followingproperties are optionally modified by inclusion of an unnatural aminoacid into a protein: toxicity, biodistribution, structural properties,spectroscopic properties, chemical and/or photochemical properties,catalytic ability, half-life (including but not limited to, serumhalf-life), ability to react with other molecules, including but notlimited to, covalently or noncovalently, and the like. The compositionsincluding proteins that include at least one unnatural amino acid areuseful for, including but not limited to, novel therapeutics,diagnostics, catalytic enzymes, industrial enzymes, binding proteins(including but not limited to, antibodies), and including but notlimited to, the study of protein structure and function. See, e.g.,Dougherty, (2000) Unnatural Amino Acids as Probes of Protein Structureand Function, Current Opinion in Chemical Biology, 4:645-652.

In one aspect of the invention, a composition includes at least oneprotein with at least one, including but not limited to, at least two,at least three, at least four, at least five, at least six, at leastseven, at least eight, at least nine, or at least ten or more unnaturalamino acids. The unnatural amino acids can be the same or different,including but not limited to, there can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or10 or more different sites in the protein that comprise 1, 2, 3, 4, 5,6, 7, 8, 9, or 10 or more different unnatural amino acids. In anotheraspect, a composition includes a protein with at least one, but fewerthan all, of a particular amino acid present in the protein issubstituted with the unnatural amino acid. For a given protein with morethan one unnatural amino acids, the unnatural amino acids can beidentical or different (including but not limited to, the protein caninclude two or more different types of unnatural amino acids, or caninclude two of the same unnatural amino acid). For a given protein withmore than two unnatural amino acids, the unnatural amino acids can bethe same, different or a combination of a multiple unnatural amino acidof the same kind with at least one different unnatural amino acid.

Proteins or polypeptides of interest with at least one unnatural aminoacid are a feature of the invention. The invention also includespolypeptides or proteins with at least one unnatural amino acid producedusing the compositions and methods of the invention. An excipient(including but not limited to, a pharmaceutically acceptable excipient)can also be present with the protein.

By producing proteins or polypeptides of interest with at least oneunnatural amino acid in eukaryotic cells, proteins or polypeptides willtypically include eukaryotic post-translational modifications. Incertain embodiments, a protein includes at least one unnatural aminoacid and at least one post-translational modification that is made invivo by a eukaryotic cell, where the post-translational modification isnot made by a prokaryotic cell. For example, the post-translationmodification includes, including but not limited to, acetylation,acylation, lipid-modification, palmitoylation, palmitate addition,phosphorylation, glycolipid-linkage modification, glycosylation, and thelike. In one aspect, the post-translational modification includesattachment of an oligosaccharide (including but not limited to,(GlcNAc-Man)₂-Man-GlcNAc-GlcNAc)) to an asparagine by aGlcNAc-asparagine linkage. See Table 1 which lists some examples ofN-linked oligosaccharides of eukaryotic proteins (additional residuescan also be present, which are not shown). In another aspect, thepost-translational modification includes attachment of anoligosaccharide (including but not limited to, Gal-GalNAc, Gal-GlcNAc,etc.) to a serine or threonine by a GalNAc-serine or GalNAc-threoninelinkage, or a GlcNAc-serine or a GlcNAc-threonine linkage.

In yet another aspect, the post-translation modification includesproteolytic processing of precursors (including but not limited to,calcitonin precursor, calcitonin gene-related peptide precursor,preproparathyroid hormone, preproinsulin, proinsulin,prepro-opiomelanocortin, pro-opiomelanocortin and the like), assemblyinto a multisubunit protein or macromolecular assembly, translation toanother site in the cell (including but not limited to, to organelles,such as the endoplasmic reticulum, the Golgi apparatus, the nucleus,lysosomes, peroxisomes, mitochondria, chloroplasts, vacuoles, etc., orthrough the secretory pathway). In certain embodiments, the proteincomprises a secretion or localization sequence, an epitope tag, a FLAGtag, a polyhistidine tag, a GST fusion, or the like.

One advantage of an unnatural amino acid is that it presents additionalchemical moieties that can be used to add additional molecules. Thesemodifications can be made in vivo in a eukaryotic or non-eukaryoticcell, or in vitro. Thus, in certain embodiments, the post-translationalmodification is through the unnatural amino acid. For example, thepost-translational modification can be through anucleophilic-electrophilic reaction. Most reactions currently used forthe selective modification of proteins involve covalent bond formationbetween nucleophilic and electrophilic reaction partners, including butnot limited to the reaction of α-haloketones with histidine or cysteineside chains. Selectivity in these cases is determined by the number andaccessibility of the nucleophilic residues in the protein. In proteinsof the invention, other more selective reactions can be used such as thereaction of an unnatural keto-amino acid with hydrazides or aminooxycompounds, in vitro and in vivo. See, e.g., Cornish, et al., (1996) J.Am. Chem. Soc., 118:8150-8151; Mahal, et al., (1997) Science,276:1125-1128; Wang, et al., (2001) Science 292:498-500; Chin, et al.,(2002) J. Am. Chem. Soc. 124:9026-9027; Chin, et al., (2002) Proc. Natl.Acad. Sci., 99:11020-11024; Wang, et al., (2003) Proc. Natl. Acad. Sci.,100:56-61; Zhang, et al., (2003) Biochemistry, 42:6735-6746; and, Chin,et al., (2003) Science, 301:964-7. This allows the selective labeling ofvirtually any protein with a host of reagents including fluorophores,crosslinking agents, saccharide derivatives and cytotoxic molecules. Seealso, U.S. patent application Ser. No. 10/686,944 entitled “Glycoproteinsynthesis” filed Oct. 15, 2003 based on U.S. provisional patentapplication Ser. No. 60/419,265, filed Oct. 16, 2002, U.S. provisionalpatent application Ser. No. 60/420,990, filed Oct. 23, 2002, and U.S.provisional patent application Ser. No. 60/441,450, filed Jan. 16, 2003,which are incorporated by reference herein. Post-translationalmodifications, including but not limited to, through an azido aminoacid, can also made through the Staudinger ligation (including but notlimited to, with triarylphosphine reagents). See, e.g., Kiick et al.,(2002) Incorporation of azides into recombinant proteins forchemoselective modification by the Staudinger ligation, PNAS 99:19-24.

This invention provides another highly efficient method for theselective modification of proteins, which involves the geneticincorporation of unnatural amino acids, including but not limited to,containing an azide or alkynyl moiety into proteins in response to aselector codon. These amino acid side chains can then be modified by,including but not limited to, a Huisgen [34-2] cycloaddition reaction(see, e.g., Padwa, A. in Comprehensive Organic Synthesis, Vol. 4, (1991)Ed. Trost, B. M., Pergamon, Oxford, p. 1069-1109; and, Huisgen, R. in1,3-Diploar Cycloaddition Chemistry, (1984) Ed. Padwa, A., Wiley, NewYork, p. 1-176) with, including but not limited to, alkynyl or azidederivatives, respectively. Because this method involves a cycloadditionrather than a nucleophilic substitution, proteins can be modified withextremely high selectivity. This reaction can be carried out at roomtemperature in aqueous conditions with excellent regioselectivity(1,4>1,5) by the addition of catalytic amounts of Cu(I) salts to thereaction mixture. See, e.g., Tornoe, et al., (2002) J. Org. Chem.67:3057-3064; and, Rostovtsev, et al., (2002) Angew. Chem. Int. Ed.41:2596-2599. Another method that can be used is the ligand exchange ona bisarsenic compound with a tetracysteine motif, see, e.g., Griffin, etal., (1998) Science 281:269-272.

A molecule that can be added to a protein of the invention through a[3+2] cycloaddition includes virtually any molecule with an azide oralkynyl derivative. Molecules include, but are not limited to, dyes,fluorophores, crosslinking agents, saccharide derivatives, polymers(including but not limited to, derivatives of polyethylene glycol),photocrosslinkers, cytotoxic compounds, affinity labels, derivatives ofbiotin, resins, beads, a second protein or polypeptide (or more),polynucleotide(s) (including but not limited to, DNA, RNA, etc.), metalchelators, cofactors, fatty acids, carbohydrates, and the like. Thesemolecules can be added to an unnatural amino acid with an alkynyl group,including but not limited to, p-propargyloxyphenylalanine, or azidogroup, including but not limited to, p-azido-phenylalanine,respectively.

V. In Vivo Generation of a BPFI Comprising Non-Genetically-Encoded AminoAcids

The BPFIs of the invention can be generated in vivo using modified tRNAand tRNA synthetases to add to or substitute amino acids that are notencoded in naturally-occurring systems.

Methods for generating tRNAs and tRNA synthetases which use amino acidsthat are not encoded in naturally-occurring systems are described in,e.g., U.S. Patent Application Publications 2003/0082575 (Ser. No.10/126,927) and 2003/0108885 (Ser. No. 10/126,931) which areincorporated by reference herein. These methods involve generating atranslational machinery that functions independently of the synthetasesand tRNAs endogenous to the translation system (and are thereforesometimes referred to as “orthogonal”). Typically, the translationsystem comprises an orthogonal tRNA (O-tRNA) and an orthogonal aminoacyltRNA synthetase (O-RS). Typically, the O-RS preferentially aminoacylatesthe O-tRNA with at least one non-naturally occurring amino acid in thetranslation system and the O-tRNA recognizes at least one selector codonthat is not recognized by other tRNAs in the system. The translationsystem thus inserts the non-naturally-encoded amino acid into a proteinproduced in the system, in response to an encoded selector codon,thereby “substituting” an amino acid into a position in the encodedpolypeptide.

A wide variety of orthogonal tRNAs and aminoacyl tRNA synthetases havebeen described in the art for inserting particular synthetic amino acidsinto polypeptides, and are generally suitable for use in the presentinvention. For example, keto-specific O-tRNA/aminoacyl-tRNA synthetasesare described in Wang, L., et al., Proc. Natl. Acad. Sci. USA 100:56-61(2003) and Zhang, Z. et al., Biochem. 42(22):6735-6746 (2003). ExemplaryO-RS, or portions thereof, are encoded by polynucleotide sequences andinclude amino acid sequences disclosed in U.S. Patent ApplicationPublications 2003/0082575 and 2003/0108885, each incorporated herein byreference. Corresponding O-tRNA molecules for use with the O-RSs arealso described in U.S. Patent Application Publications 2003/0082575(Ser. No. 10/126,927) and 2003/0108885 (Ser. No. 10/126,931) which areincorporated by reference herein.

An example of an azide-specific O-tRNA/aminoacyl-tRNA synthetase systemis described in Chin, J. W., et al., J. Am. Chem. Soc. 124:9026-9027(2002). Exemplary O-RS sequences for p-azido-L-Phe include, but are notlimited to, nucleotide sequences SEQ ID NOs: 14-16 and 29-32 and aminoacid sequences SEQ ID NOs: 46-48 and 61-64 as disclosed in U.S. PatentApplication Publication 2003/0108885 (Ser. No. 10/126,931) which isincorporated by reference herein. Exemplary O-tRNA sequences suitablefor use in the present invention include, but are not limited to,nucleotide sequences SEQ ID NOs: 1-3 as disclosed in U.S. PatentApplication Publication 2003/0108885 (Ser. No. 10/126,931) which isincorporated by reference herein. Other examples ofO-tRNA/aminoacyl-tRNA synthetase pairs specific to particularnon-naturally encoded amino acids are described in U.S. PatentApplication Publication 2003/0082575 (Ser. No. 10/126,927) which isincorporated by reference herein. O-RS and O-tRNA that incorporate bothketo- and azide-containing amino acids in S. cerevisiae are described inChin, J. W., et al., Science 301:964-967 (2003).

Use of O-tRNA/aminoacyl-tRNA synthetases involves selection of aspecific codon which encodes the non-naturally encoded amino acid. Whileany codon can be used, it is generally desirable to select a codon thatis rarely or never used in the cell in which the O-tRNA/aminoacyl-tRNAsynthetase is expressed. For example, exemplary codons include nonsensecodon such as stop codons (amber, ochre, and opal), four or more basecodons and other natural three-base codons that are rarely or unused.

Specific selector codon(s) can be introduced into appropriate positionsin the BPFI coding sequence using mutagenesis methods known in the art(including but not limited to, site-specific mutagenesis, cassettemutagenesis, restriction selection mutagenesis, etc.).

Methods for generating components of the protein biosynthetic machinery,such as O-RSs, O-tRNAs, and orthogonal O-tRNA/O-RS pairs that can beused to incorporate a non-naturally encoded amino acid are described inWang, L., et al., Science 292: 498-500 (2001); Chin, J. W., et al., J.Am. Chem. Soc. 124:9026-9027 (2002); Zhang, Z. et al., Biochemistry 42:6735-6746 (2003). Methods and compositions for the in vivo incorporationof non-naturally encoded amino acids are described in U.S. PatentApplication Publication 2003/0082575 (Ser. No. 10/126,927) which isincorporated by reference herein. Methods for selecting an orthogonaltRNA-tRNA synthetase pair for use in in vivo translation system of anorganism are also described in U.S. Patent Application Publications2003/0082575 (Ser. No. 10/126,927) and 2003/0108885 (Ser. No.10/126,931) which are incorporated by reference herein.

Methods for producing at least one recombinant orthogonal aminoacyl-tRNAsynthetase (O-RS) comprise: (a) generating a library of (optionallymutant) RSs derived from at least one aminoacyl-tRNA synthetase (RS)from a first organism, including but not limited to, a prokaryoticorganism, such as Methanococcus jannaschii, Methanobacteriumthermoautotrophicum, Halobacterium, Escherichia coli, A. fulgidus, P.furiosus, P. horikoshii, A. pernix, T thermophilus, or the like, or aeukaryotic organism; (b) selecting (and/or screening) the library of RSs(optionally mutant RSs) for members that aminoacylate an orthogonal tRNA(O-tRNA) in the presence of a non-naturally encoded amino acid and anatural amino acid, thereby providing a pool of active (optionallymutant) RSs; and/or, (c) selecting (optionally through negativeselection) the pool for active RSs (including but not limited to, mutantRSs) that preferentially aminoacylate the O-tRNA in the absence of thenon-naturally encoded amino acid, thereby providing the at least onerecombinant O-RS; wherein the at least one recombinant O-RSpreferentially aminoacylates the O-tRNA with the non-naturally encodedamino acid.

In one embodiment, the RS is an inactive RS. The inactive RS can begenerated by mutating an active RS. For example, the inactive RS can begenerated by mutating at least about 1, at least about 2, at least about3, at least about 4, at least about 5, at least about 6, or at leastabout 10 or more amino acids to different amino acids, including but notlimited to, alanine.

Libraries of mutant RSs can be generated using various techniques knownin the art, including but not limited to rational design based onprotein three dimensional RS structure, or mutagenesis of RS nucleotidesin a random or rational design technique. For example, the mutant RSscan be generated by site-specific mutations, random mutations, diversitygenerating recombination mutations, chimeric constructs, rational designand by other methods described herein or known in the art.

In one embodiment, selecting (and/or screening) the library of RSs(optionally mutant RSs) for members that are active, including but notlimited to, that aminoacylate an orthogonal tRNA (O-tRNA) in thepresence of a non-naturally encoded amino acid and a natural amino acid,includes: introducing a positive selection or screening marker,including but not limited to, an antibiotic resistance gene, or thelike, and the library of (optionally mutant) RSs into a plurality ofcells, wherein the positive selection and/or screening marker comprisesat least one selector codon, including but not limited to, an amber,ochre, or opal codon; growing the plurality of cells in the presence ofa selection agent; identifying cells that survive (or show a specificresponse) in the presence of the selection and/or screening agent bysuppressing the at least one selector codon in the positive selection orscreening marker, thereby providing a subset of positively selectedcells that contains the pool of active (optionally mutant) RSs.Optionally, the selection and/or screening agent concentration can bevaried.

In one aspect, the positive selection marker is a chloramphenicolacetyltransferase (CAT) gene and the selector codon is an amber stopcodon in the CAT gene. Optionally, the positive selection marker is aβ-lactamase gene and the selector codon is an amber stop codon in theβ-lactamase gene. In another aspect the positive screening markercomprises a fluorescent or luminescent screening marker or an affinitybased screening marker (including but not limited to, a cell surfacemarker).

In one embodiment, negatively selecting or screening the pool for activeRSs (optionally mutants) that preferentially aminoacylate the O-tRNA inthe absence of the non-naturally encoded amino acid includes:introducing a negative selection or screening marker with the pool ofactive (optionally mutant) RSs from the positive selection or screeninginto a plurality of cells of a second organism, wherein the negativeselection or screening marker comprises at least one selector codon(including but not limited to, an antibiotic resistance gene, includingbut not limited to, a chloramphenicol acetyltransferase (CAT) gene);and, identifying cells that survive or show a specific screeningresponse in a first medium supplemented with the non-naturally encodedamino acid and a screening or selection agent, but fail to survive or toshow the specific response in a second medium not supplemented with thenon-naturally encoded amino acid and the selection or screening agent,thereby providing surviving cells or screened cells with the at leastone recombinant O-RS. For example, a CAT identification protocoloptionally acts as a positive selection and/or a negative screening indetermination of appropriate O-RS recombinants. For instance, a pool ofclones is optionally replicated on growth plates containing CAT (whichcomprises at least one selector codon) either with or without one ormore non-naturally encoded amino acid. Colonies growing exclusively onthe plates containing non-naturally encoded amino acids are thusregarded as containing recombinant O-RS. In one aspect, theconcentration of the selection (and/or screening) agent is varied. Insome aspects the first and second organisms are different. Thus, thefirst and/or second organism optionally comprises: a prokaryote, aeukaryote, a mammal, an Escherichia coli, a fungi, a yeast, anarchaebacterium, a eubacterium, a plant, an insect, a protist, etc. Inother embodiments, the screening marker comprises a fluorescent orluminescent screening marker or an affinity based screening marker.

In another embodiment, screening or selecting (including but not limitedto, negatively selecting) the pool for active (optionally mutant) RSsincludes: isolating the pool of active mutant RSs from the positiveselection step (b); introducing a negative selection or screeningmarker, wherein the negative selection or screening marker comprises atleast one selector codon (including but not limited to, a toxic markergene, including but not limited to, a ribonuclease barnase gene,comprising at least one selector codon), and the pool of active(optionally mutant) RSs into a plurality of cells of a second organism;and identifying cells that survive or show a specific screening responsein a first medium not supplemented with the non-naturally encoded aminoacid, but fail to survive or show a specific screening response in asecond medium supplemented with the non-naturally encoded amino acid,thereby providing surviving or screened cells with the at least onerecombinant O-RS, wherein the at least one recombinant O-RS is specificfor the non-naturally encoded amino acid. In one aspect, the at leastone selector codon comprises about two or more selector codons. Suchembodiments optionally can include wherein the at least one selectorcodon comprises two or more selector codons, and wherein the first andsecond organism are different (including but not limited to, eachorganism is optionally, including but not limited to, a prokaryote, aeukaryote, a mammal, an Escherichia coli, a fungi, a yeast, anarchaebacteria, a eubacteria, a plant, an insect, a protist, etc.).Also, some aspects include wherein the negative selection markercomprises a ribonuclease barnase gene (which comprises at least oneselector codon). Other aspects include wherein the screening markeroptionally comprises a fluorescent or luminescent screening marker or anaffinity based screening marker. In the embodiments herein, thescreenings and/or selections optionally include variation of thescreening and/or selection stringency.

In one embodiment, the methods for producing at least one recombinantorthogonal aminoacyl-tRNA synthetase (O-RS) can further comprise: (d)isolating the at least one recombinant O-RS; (e) generating a second setof O-RS (optionally mutated) derived from the at least one recombinantO-RS; and, (f) repeating steps (b) and (c) until a mutated O-RS isobtained that comprises an ability to preferentially aminoacylate theO-tRNA. Optionally, steps (d)-(f) are repeated, including but notlimited to, at least about two times. In one aspect, the second set ofmutated O-RS derived from at least one recombinant O-RS can be generatedby mutagenesis, including but not limited to, random mutagenesis,site-specific mutagenesis, recombination or a combination thereof.

The stringency of the selection/screening steps, including but notlimited to, the positive selection/screening step (b), the negativeselection/screening step (c) or both the positive and negativeselection/screening steps (b) and (c), in the above-described methods,optionally includes varying the selection/screening stringency. Inanother embodiment, the positive selection/screening step (b), thenegative selection/screening step (c) or both the positive and negativeselection/screening steps (b) and (c) comprise using a reporter, whereinthe reporter is detected by fluorescence-activated cell sorting (FACS)or wherein the reporter is detected by luminescence. Optionally, thereporter is displayed on a cell surface, on a phage display or the likeand selected based upon affinity or catalytic activity involving thenon-naturally encoded amino acid or an analogue. In one embodiment, themutated synthetase is displayed on a cell surface, on a phage display orthe like.

Methods for producing a recombinant orthogonal tRNA (O-tRNA) include:(a) generating a library of mutant tRNAs derived from at least one tRNA,including but not limited to, a suppressor tRNA, from a first organism;(b) selecting (including but not limited to, negatively selecting) orscreening the library for (optionally mutant) tRNAs that areaminoacylated by an aminoacyl-tRNA synthetase (RS) from a secondorganism in the absence of a RS from the first organism, therebyproviding a pool of tRNAs (optionally mutant); and, (c) selecting orscreening the pool of tRNAs (optionally mutant) for members that areaminoacylated by an introduced orthogonal RS (O-RS), thereby providingat least one recombinant O-tRNA; wherein the at least one recombinantO-tRNA recognizes a selector codon and is not efficiency recognized bythe RS from the second organism and is preferentially aminoacylated bythe O-RS. In some embodiments the at least one tRNA is a suppressor tRNAand/or comprises a unique three base codon of natural and/or unnaturalbases, or is a nonsense codon, a rare codon, an unnatural codon, a codoncomprising at least 4 bases, an amber codon, an ochre codon, or an opalstop codon. In one embodiment, the recombinant O-tRNA possesses animprovement of orthogonality. It will be appreciated that in someembodiments, O-tRNA is optionally imported into a first organism from asecond organism without the need for modification. In variousembodiments, the first and second organisms are either the same ordifferent and are optionally chosen from, including but not limited to,prokaryotes (including but not limited to, Methanococcus jannaschii,Methanobacteium thermoautotrophicum, Escherichia coli, Halobacterium,etc.), eukaryotes, mammals, fungi, yeasts, archaebacteria, eubacteria,plants, insects, protists, etc. Additionally, the recombinant tRNA isoptionally aminoacylated by a non-naturally encoded amino acid, whereinthe non-naturally encoded amino acid is biosynthesized in vivo eithernaturally or through genetic manipulation. The non-naturally encodedamino acid is optionally added to a growth medium for at least the firstor second organism.

In one aspect, selecting (including but not limited to, negativelyselecting) or screening the library for (optionally mutant) tRNAs thatare aminoacylated by an aminoacyl-tRNA synthetase (step (b)) includes:introducing a toxic marker gene, wherein the toxic marker gene comprisesat least one of the selector codons (or a gene that leads to theproduction of a toxic or static agent or a gene essential to theorganism wherein such marker gene comprises at least one selector codon)and the library of (optionally mutant) tRNAs into a plurality of cellsfrom the second organism; and, selecting surviving cells, wherein thesurviving cells contain the pool of (optionally mutant) tRNAs comprisingat least one orthogonal tRNA or nonfunctional tRNA. For example,surviving cells can be selected by using a comparison ratio cell densityassay.

In another aspect, the toxic marker gene can include two or moreselector codons. In another embodiment of the methods, the toxic markergene is a ribonuclease barnase gene, where the ribonuclease barnase genecomprises at least one amber codon. Optionally, the ribonuclease barnasegene can include two or more amber codons.

In one embodiment, selecting or screening the pool of (optionallymutant) tRNAs for members that are aminoacylated by an introducedorthogonal RS (O-RS) can include: introducing a positive selection orscreening marker gene, wherein the positive marker gene comprises a drugresistance gene (including but not limited to, β-lactamase gene,comprising at least one of the selector codons, such as at least oneamber stop codon) or a gene essential to the organism, or a gene thatleads to detoxification of a toxic agent, along with the O-RS, and thepool of (optionally mutant) tRNAs into a plurality of cells from thesecond organism; and, identifying surviving or screened cells grown inthe presence of a selection or screening agent, including but notlimited to, an antibiotic, thereby providing a pool of cells possessingthe at least one recombinant tRNA, where the at least one recombinanttRNA is aminoacylated by the O-RS and inserts an amino acid into atranslation product encoded by the positive marker gene, in response tothe at least one selector codons. In another embodiment, theconcentration of the selection and/or screening agent is varied.

Methods for generating specific O-tRNA/O-RS pairs are provided. Methodsinclude: (a) generating a library of mutant tRNAs derived from at leastone tRNA from a first organism; (b) negatively selecting or screeningthe library for (optionally mutant) tRNAs that are aminoacylated by anaminoacyl-tRNA synthetase (RS) from a second organism in the absence ofa RS from the first organism, thereby providing a pool of (optionallymutant) tRNAs; (c) selecting or screening the pool of (optionallymutant) tRNAs for members that are aminoacylated by an introducedorthogonal RS (O-RS), thereby providing at least one recombinant O-tRNA.The at least one recombinant O-tRNA recognizes a selector codon and isnot efficiency recognized by the RS from the second organism and ispreferentially aminoacylated by the O-RS. The method also includes (d)generating a library of (optionally mutant) RSs derived from at leastone aminoacyl-tRNA synthetase (RS) from a third organism; (e) selectingor screening the library of mutant RSs for members that preferentiallyaminoacylate the at least one recombinant O-tRNA in the presence of anon-naturally encoded amino acid and a natural amino acid, therebyproviding a pool of active (optionally mutant) RSs; and, (f) negativelyselecting or screening the pool for active (optionally mutant) RSs thatpreferentially aminoacylate the at least one recombinant O-tRNA in theabsence of the non-naturally encoded amino acid, thereby providing theat least one specific O-tRNA/O-RS pair, wherein the at least onespecific O-tRNA/O-RS pair comprises at least one recombinant O-RS thatis specific for the non-naturally encoded amino acid and the at leastone recombinant O-tRNA. Specific O-tRNA/O-RS pairs produced by themethods are included. For example, the specific O-tRNA/O-RS pair caninclude, including but not limited to, a mutRNATyr-mutTyrRS pair, suchas a mutRNATyr-SS12TyrRS pair, a mutRNALeu-mutLeuRS pair, amutRNAThr-mutThrRS pair, a mutRNAGlu-mutGluRS pair, or the like.Additionally, such methods include wherein the first and third organismare the same (including but not limited to, Methanococcus jannaschii).

Methods for selecting an orthogonal tRNA-tRNA synthetase pair for use inan in vivo translation system of a second organism are also included inthe present invention. The methods include: introducing a marker gene, atRNA and an aminoacyl-tRNA synthetase (RS) isolated or derived from afirst organism into a first set of cells from the second organism;introducing the marker gene and the tRNA into a duplicate cell set froma second organism; and, selecting for surviving cells in the first setthat fail to survive in the duplicate cell set or screening for cellsshowing a specific screening response that fail to give such response inthe duplicate cell set, wherein the first set and the duplicate cell setare grown in the presence of a selection or screening agent, wherein thesurviving or screened cells comprise the orthogonal tRNA-tRNA synthetasepair for use in the in the in vivo translation system of the secondorganism. In one embodiment, comparing and selecting or screeningincludes an in vivo complementation assay. The concentration of theselection or screening agent can be varied.

The organisms of the present invention comprise a variety of organismand a variety of combinations. For example, the first and the secondorganisms of the methods of the present invention can be the same ordifferent. In one embodiment, the organisms are optionally a prokaryoticorganism, including but not limited to, Methanococcus jannaschii,Methanobacterium thermoautotrophicum, Halobacterium, Escherichia coli,A. fulgidus, P. furiosus, P. horikoshii, A. pernix, T thermophilus, orthe like. Alternatively, the organisms optionally comprise a eukaryoticorganism, including but not limited to, plants (including but notlimited to, complex plants such as monocots, or dicots), algae,protists, fungi (including but not limited to, yeast, etc), animals(including but not limited to, mammals, insects, arthropods, etc.), orthe like. In another embodiment, the second organism is a prokaryoticorganism, including but not limited to, Methanococcus jannaschii,Methanobacterium thermoautotrophicum, Halobacterium, Escherichia coli,A. fulgidus, Halobacterium, P. furiosus, P. horikoshii, A. pernix, Tthermophilus, or the like. Alternatively, the second organism can be aeukaryotic organism, including but not limited to, a yeast, a animalcell, a plant cell, a fungus, a mammalian cell, or the like. In variousembodiments the first and second organisms are different.

VI. Location of Non-Naturally-Occurring Amino Acids in a BPFI

The present invention contemplates incorporation of one or morenon-naturally-occurring amino acids into a BPFI. One or morenon-naturally-occurring amino acids may be incorporated at a particularposition which does not disrupt activity of the polypeptide. This can beachieved by making “conservative” substitutions, including but notlimited to, substituting hydrophobic amino acids with hydrophobic aminoacids, bulky amino acids for bulky amino acids, hydrophilic amino acidsfor hydrophilic amino acids) and/or inserting thenon-naturally-occurring amino acid in a location that is not requiredfor activity.

A variety of biochemical and structural approaches can be employed toselect the desired sites for substitution with a non-naturally encodedamino acid within the BPFI. It is readily apparent to those of ordinaryskill in the art that any position of the polypeptide chain is suitablefor selection to incorporate a non-naturally encoded amino acid, andselection may be based on rational design or by random selection for anyor no particular desired purpose. Selection of desired sites may be forproducing a BPFI molecule having any desired property or activity,including but not limited to, agonists, super-agonists, inverseagonists, antagonists, receptor binding modulators, receptor activitymodulators, modulators of binding to binding partners, binding partneractivity modulators, binding partner conformation modulators, dimer ormultimer formation, no change to activity or property compared to thenative molecule, or manipulating any physical or chemical property ofthe polypeptide such as solubility, aggregation, or stability. Forexample, locations in the polypeptide required for biological activityof BPFI can be identified using point mutation analysis, alaninescanning or homolog scanning methods known in the art. Residues otherthan those identified as critical to biological activity by alanine orhomolog scanning mutagenesis may be good candidates for substitutionwith a non-naturally encoded amino acid depending on the desiredactivity sought for the polypeptide. Alternatively, the sites identifiedas critical to biological activity may also be good candidates forsubstitution with a non-naturally encoded amino acid, again depending onthe desired activity sought for the polypeptide. Another alternativewould be to simply make serial substitutions in each position on thepolypeptide chain with a non-naturally encoded amino acid and observethe effect on the activities of the polypeptide. It is readily apparentto those of ordinary skill in the art that any means, technique, ormethod for selecting a position for substitution with a non-naturalamino acid into any polypeptide is suitable for use in the presentinvention.

The structure and activity of naturally-occurring mutants of BPFI thatcontain deletions can also be examined to determine regions of theprotein that are likely to be tolerant of substitution with anon-naturally encoded amino acid. In a similar manner, proteasedigestion and monoclonal antibodies can be used to identify regions ofBPFI that are responsible for binding the BPFI receptor or bindingpartners. Once residues that are likely to be intolerant to substitutionwith non-naturally encoded amino acids have been eliminated, the impactof proposed substitutions at each of the remaining positions can beexamined from the structure of BPFI and its receptor or bindingpartners. Thus, those of skill in the art can readily identify aminoacid positions that can be substituted with non-naturally encoded aminoacids.

In some embodiments, the BPFIs of the invention comprise one or morenon-naturally occurring amino acids positioned in a region of theprotein that does not disrupt the helices or beta sheet secondarystructure of the polypeptide.

In some embodiments, one or more non-naturally encoded amino acids areincorporated at any position in HR-N, HR-C or anionic peptide, beforethe first amino acid (at the amino terminus), an addition at the carboxyterminus, or any combination thereof.

In some embodiments, the non-naturally occurring amino acid at these orother positions is linked to a water soluble molecule.

Exemplary residues of incorporation of a non-naturally encoded aminoacid may be those that are included or excluded from potential receptorbinding regions or regions for binding to binding partners, may be fullyor partially solvent exposed, have minimal or no hydrogen-bondinginteractions with nearby residues, may be minimally exposed to nearbyreactive residues, may be on one or more of the exposed faces of theBPFI, may be in regions that are highly flexible, or structurally rigid,as predicted by the three-dimensional, secondary, tertiary, orquaternary structure of the BPFI, bound or unbound to its receptor orbinding partner, or coupled or not coupled to another BPFI or otherbiologically active molecule, or may modulate the conformation of theBPFI itself or a dimer or multimer comprising one or more BPFI, byaltering the flexibility or rigidity of the complete structure asdesired.

In one embodiment, the method further includes incorporating into theprotein the unnatural amino acid, where the unnatural amino acidcomprises a first reactive group; and contacting the protein with amolecule (including but not limited to, a label, a dye, a polymer, awater-soluble polymer, a derivative of polyethylene glycol, aphotocrosslinker, a radionuclide, a cytotoxic compound, a drug, anaffinity label, a photoaffinity label, a reactive compound, a resin, asecond protein or polypeptide or polypeptide analog, an antibody orantibody fragment, a metal chelator, a cofactor, a fatty acid, acarbohydrate, a polynucleotide, a DNA, a RNA, an antisensepolynucleotide, a water soluble dendimer, a cyclodextrin, an inhibitoryribonucleic acid, a biomaterial, a nanoparticle, a spin label, afluorophore, a metal-containing moiety, a radioactive moiety, a novelfunctional group, a group that covalently or noncovalently interactswith other molecules, a photocaged moiety, a photoisomerizable moiety,biotin, a derivative of biotin, a biotin analogue, a moietyincorporating a heavy atom, a chemically cleavable group, aphotocleavable group, an elongated side chain, a carbon-linked sugar, aredox-active agent, an amino thioacid, a toxic moiety, an isotopicallylabeled moiety, a biophysical probe, a phosphorescent group, achemiluminescent group, an electron dense group, a magnetic group, anintercalating group, a chromophore, an energy transfer agent, abiologically active agent, a detectable label, a small molecule, or anycombination of the above, or any other desirable compound or substance)that comprises a second reactive group. The first reactive group reactswith the second reactive group to attach the molecule to the unnaturalamino acid through a [3+2] cycloaddition. In one embodiment, the firstreactive group is an alkynyl or azido moiety and the second reactivegroup is an azido or alkynyl moiety. For example, the first reactivegroup is the alkynyl moiety (including but not limited to, in unnaturalamino acid p-propargyloxyphenylalanine) and the second reactive group isthe azido moiety. In another example, the first reactive group is theazido moiety (including but not limited to, in the unnatural amino acidp-azido-L-phenylalanine) and the second reactive group is the alkynylmoiety.

In some cases, the non-naturally encoded amino acid substitution(s) willbe combined with other additions, substitutions or deletions within theBPFI to affect other biological traits of the BPFI. In some cases, theother additions, substitutions or deletions may increase the stability(including but not limited to, resistance to proteolytic degradation) ofthe BPFI or increase affinity of the BPFI for its receptor or bindingpartner. In some cases, the other additions, substitutions or deletionsmay increase the solubility (including but not limited to, whenexpressed in E. coli or other host cells) of the BPFI. In someembodiments additions, substitutions or deletions may increase thepolypeptide solubility following expression in E. coli recombinant hostcells. In some embodiments sites are selected for substitution with anaturally encoded or non-natural amino acid in addition to another sitefor incorporation of a non-natural amino acid that results in increasingthe polypeptide solubility following expression in E. coli recombinanthost cells. In some embodiments, the BPFIs comprise another addition,substitution or deletion that modulates affinity for the BPFI receptoror binding partner, modulates (including but not limited to, increasesor decreases) receptor dimerization, stabilizes receptor dimers,modulates the conformation or one or biological activites of a bindingpartner, modulates circulating half-life, modulates release orbio-availability, facilitates purification, or improves or alters aparticular route of administration. Similarly, BPFIs can compriseprotease cleavage sequences, reactive groups, antibody-binding domains(including but not limited to, FLAG or poly-His) or other affinity basedsequences (including, but not limited to, FLAG, poly-His, GST, etc.) orlinked molecules (including, but not limited to, biotin) that improvedetection (including, but not limited to, GFP), purification, transportthrough tissues or cell membranes, prodrug release or activation, BPFIsize reduction, or other traits of the polypeptide.

VII. Expression in Non-Eukaryotes and Eukaryotes

To obtain high level expression of a cloned BPFI, one typicallysubclones polynucleotides encoding a BPFI of the invention into anexpression vector that contains a strong promoter to directtranscription, a transcription/translation terminator, and if for anucleic acid encoding a protein, a ribosome binding site fortranslational initiation. Suitable bacterial promoters are well known inthe art and described, e.g., in Sambrook et al. and Ausubel et al. Asuitable strategy for constructing an expression vector for expressionof a BPFI of the present invention includes, but is not limited to thestrategy shown in FIG. 2.

Bacterial expression systems for expressing BPFIs of the invention areavailable in, including but not limited to, E. coli, Bacillus sp., andSalmonella (Palva et al., Gene 22:229-235 (1983); Mosbach et al., Nature302:543-545 (1983)). Kits for such expression systems are commerciallyavailable. Eukaryotic expression systems for mammalian cells, yeast, andinsect cells are well known in the art and are also commerciallyavailable. In cases where orthogonal tRNAs and aminoacyl tRNAsynthetases (described above) are used to express the BPFIs of theinvention, host cells for expression are selected based on their abilityto use the orthogonal components. Exemplary host cells includeGram-positive bacteria (including but not limited to B. brevis, B.subtilis, or Streptomyces) and Gram-negative bacteria (E. coli,Pseudomonas fluorescens, Pseudomonas aeruginosa, Pseudomonas putida), aswell as yeast and other eukaryotic cells. Cells comprising O-tRNA/O-RSpairs can be used as described herein.

A eukaryotic host cell or non-eukaryotic host cell of the presentinvention provides the ability to synthesize proteins that compriseunnatural amino acids in large useful quantities. In one aspect, thecomposition optionally includes, including but not limited to, at least10 micrograms, at least 50 micrograms, at least 75 micrograms, at least100 micrograms, at least 200 micrograms, at least 250 micrograms, atleast 500 micrograms, at least 1 milligram, at least 10 milligrams, atleast 100 milligrams, at least one gram, or more of the protein thatcomprises an unnatural amino acid, or an amount that can be achievedwith in vivo protein production methods (details on recombinant proteinproduction and purification are provided herein). In another aspect, theprotein is optionally present in the composition at a concentration of,including but not limited to, at least 10 micrograms of protein perliter, at least 50 micrograms of protein per liter, at least 75micrograms of protein per liter, at least 100 micrograms of protein perliter, at least 200 micrograms of protein per liter, at least 250micrograms of protein per liter, at least 500 micrograms of protein perliter, at least 1 milligram of protein per liter, or at least 10milligrams of protein per liter or more, in, including but not limitedto, a cell lysate, a buffer, a pharmaceutical buffer, or other liquidsuspension (including but not limited to, in a volume of, including butnot limited to, anywhere from about 1 nl to about 100 L). The productionof large quantities (including but not limited to, greater that thattypically possible with other methods, including but not limited to, invitro translation) of a protein in a eukaryotic cell including at leastone unnatural amino acid is a feature of the invention.

A eukaryotic host cell or non-eukaryotic host cell of the presentinvention provides the ability to biosynthesize proteins that compriseunnatural amino acids in large useful quantities. For example, proteinscomprising an unnatural amino acid can be produced at a concentrationof, including but not limited to, at least 10 μg/liter, at least 50μg/liter, at least 75 μg/liter, at least 100 μg/liter, at least 200μg/liter, at least 250 μg/liter, or at least 500 μg/liter, at least 1mg/liter, at least 2 mg/liter, at least 3 mg/liter, at least 4 mg/liter,at least 5 mg/liter, at least 6 mg/liter, at least 7 mg/liter, at least8 mg/liter, at least 9 mg/liter, at least 10 mg/liter, at least 20, 30,40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900mg/liter, 1 g/liter, 5 g/liter, 10 g/liter or more of protein in a cellextract, cell lysate, culture medium, a buffer, and/or the like.

I. Expression Systems, Culture, and Isolation

BPFIs may be expressed in any number of suitable expression systemsincluding, for example, yeast, insect cells, mammalian cells, andbacteria. A description of exemplary expression systems is providedbelow.

Yeast As used herein, the term “yeast” includes any of the variousyeasts capable of expressing a gene encoding a BPFI. Such yeastsinclude, but are not limited to, ascosporogenous yeasts (Endomycetales),basidiosporogenous yeasts and yeasts belonging to the Fungi imperfecti(Blastomycetes) group. The ascosporogenous yeasts are divided into twofamilies, Spermophthoraceae and Saccharomycetaceae. The latter iscomprised of four subfamilies, Schizosaccharomycoideae (e.g., genusSchizosaccharomyces), Nadsonioideae, Lipomycoideae and Saccharomycoideae(e.g., genera Pichia, Kluyveromyces and Saccharomyces). Thebasidiosporogenous yeasts include the genera Leucosporidium,Rhodosporidium, Sporidiobolus, Filobasidium, and Filobasidiella. Yeastsbelonging to the Fungi Imperfecti (Blastomycetes) group are divided intotwo families, Sporobolomycetaceae (e.g., genera Sporobolomyces andBullera) and Cryptococcaceae (e.g., genus Candida).

Of particular interest for use with the present invention are specieswithin the genera Pichia, Kluyveromyces, Saccharomyces,Schizosaccharomyces, Hansenula, Torulopsis, and Candida, including, butnot limited to, P. pastoris, P. guillerimondii, S. cerevisiae, S.carlsbergensis, S. diastaticus, S. douglasii, S. kluyveri, S, norbensis,S. oviformis, K lactis, K fragilis, C. albicans, C. maltosa, and H.polymorpha.

The selection of suitable yeast for expression of BPFI is within theskill of one of ordinary skill in the art. In selecting yeast hosts forexpression, suitable hosts may include those shown to have, for example,good secretion capacity, low proteolytic activity, good soluble proteinproduction, and overall robustness. Yeast are generally available from avariety of sources including, but not limited to, the Yeast GeneticStock Center, Department of Biophysics and Medical Physics, Universityof California (Berkeley, Calif.), and the American Type CultureCollection (“ATCC”) (Manassas, Va.).

The term “yeast host” or “yeast host cell” includes yeast that can be,or has been, used as a recipient for recombinant vectors or othertransfer DNA. The term includes the progeny of the original yeast hostcell that has received the recombinant vectors or other transfer DNA. Itis understood that the progeny of a single parental cell may notnecessarily be completely identical in morphology or in genomic or totalDNA complement to the original parent, due to accidental or deliberatemutation. Progeny of the parental cell that are sufficiently similar tothe parent to be characterized by the relevant property, such as thepresence of a nucleotide sequence encoding BPFI, are included in theprogeny intended by this definition.

Expression and transformation vectors, including extrachromosomalreplicons or integrating vectors, have been developed for transformationinto many yeast hosts. For example, expression vectors have beendeveloped for S. cerevisiae (Sikorski et al., GENETICS (1989) 122:19;Ito et al., J. BACTERIOL. (1983) 153:163; Hinnen et al., PROC. NATL.ACAD. SCI. USA (1978) 75:1929); C. albicans (Kurtz et al., MOL. CELL.BIOL. (1986) 6:142); C. maltoso (Kunze et al., J. BASIC MICROBIOL.(1985) 25:141); H. polymorpha (Gleeson et al., J. GEN. MICROBIOL. (1986)132:3459; Roggenkamp et al., MOL. GENETICS AND GENOMICS (1986) 202:302);K. fragilis (Das et al., J. BACTERIOL. (1984) 158:1165); K. lactis (DeLouvencourt et al., J. BACTERIOL. (1983) 154:737; Van den Berg et al.,BIOTECHNOLOGY (NY) (1990) 8:135); P. guillerimondii (Kunze et al., J.BASIC MICROBIOL. (1985) 25:141); P. pastoris (U.S. Pat. Nos. 5,324,639;4,929,555; and 4,837,148; Cregg et al., MOL. CELL. BIOL. (1985) 5:3376);Schizosaccharomyces pombe (Beach et al., NATURE (1982) 300:706); and Y.lipolytica (Davidow et al., CURR. GENET. (1985) 10:380 (1985);Gaillardin et al., CURR. GENET. (1986) 10:49); A. nidulans (Ballance etal., BIOCHEM. BIOPHYS. RES. COMMUN. (1983) 112:284-89; Tilburn et al.,GENE (1983) 26:205-221; and Yelton et al., PROC. NATL. ACAD. SCI. USA(1984) 81:1470-74); A. niger (Kelly and Hynes, EMBO J. (1985)4:475-479); T. reesia (EP 0 244 234); and filamentous fungi such as,e.g., Neurospora, Penicillium, Tolypocladium (WO 91/00357), eachincorporated by reference herein.

Control sequences for yeast vectors are well known to those of ordinaryskill in the art and include, but are not limited to, promoter regionsfrom genes such as alcohol dehydrogenase (ADH) (EP 0 284 044); enolase;glucokinase; glucose-6-phosphate isomerase;glyceraldehydes-3-phosphate-dehydrogenase (GAP or GAPDH); hexokinase;phosphofructokinase; 3-phosphoglycerate mutase; and pyruvate kinase(PyK) (EP 0 329 203). The yeast PHO5 gene, encoding acid phosphatase,also may provide useful promoter sequences (Miyanohara et al., PROC.NATL. ACAD. SCI. USA (1983) 80:1). Other suitable promoter sequences foruse with yeast hosts may include the promoters for 3-phosphoglyceratekinase (Hitzeman et al., J. BIOL. CHEM. (1980) 255:12073); and otherglycolytic enzymes, such as pyruvate decarboxylase, triosephosphateisomerase, and phosphoglucose isomerase (Holland et al., BIOCHEMISTRY(1978) 17:4900; Hess et al., J. ADV. ENZYME REG. (1969) 7:149).Inducible yeast promoters having the additional advantage oftranscription controlled by growth conditions may include the promoterregions for alcohol dehydrogenase 2; isocytochrome C; acid phosphatase;metallothionein; glyceraldehyde-3-phosphate dehydrogenase; degradativeenzymes associated with nitrogen metabolism; and enzymes responsible formaltose and galactose utilization. Suitable vectors and promoters foruse in yeast expression are further described in EP 0 073 657.

Yeast enhancers also may be used with yeast promoters. In addition,synthetic promoters may also function as yeast promoters. For example,the upstream activating sequences (UAS) of a yeast promoter may bejoined with the transcription activation region of another yeastpromoter, creating a synthetic hybrid promoter. Examples of such hybridpromoters include the ADH regulatory sequence linked to the GAPtranscription activation region. See U.S. Pat. Nos. 4,880,734 and4,876,197, which are incorporated by reference herein. Other examples ofhybrid promoters include promoters that consist of the regulatorysequences of the ADH2, GAL4, GAL10, or PHO5 genes, combined with thetranscriptional activation region of a glycolytic enzyme gene such asGAP or PyK. See EP 0 164 556. Furthermore, a yeast promoter may includenaturally occurring promoters of non-yeast origin that have the abilityto bind yeast RNA polymerase and initiate transcription.

Other control elements that may comprise part of the yeast expressionvectors include terminators, for example, from GAPDH or the enolasegenes (Holland et al., J. BIOL. CHEM. (1981) 256:1385). In addition, theorigin of replication from the 2μ plasmid origin is suitable for yeast.A suitable selection gene for use in yeast is the trp1 gene present inthe yeast plasmid. See Tschumper et al., GENE (1980) 10:157; Kingsman etal., GENE (1979) 7:141. The trp1 gene provides a selection marker for amutant strain of yeast lacking the ability to grow in tryptophan.Similarly, Leu2-deficient yeast strains (ATCC 20,622 or 38,626) arecomplemented by known plasmids bearing the Leu2 gene.

Methods of introducing exogenous DNA into yeast hosts are well known tothose of ordinary skill in the art, and typically include, but are notlimited to, either the transformation of spheroplasts or of intact yeasthost cells treated with alkali cations. For example, transformation ofyeast can be carried out according to the method described in Hsiao etal., PROC. NATL. ACAD. SCI. USA (1979) 76:3829 and Van Solingen et al.,J. BACT. (1977) 130:946. However, other methods for introducing DNA intocells such as by nuclear injection, electroporation, or protoplastfusion may also be used as described generally in SAMBROOK ET AL.,MOLECULAR CLONING: A LAB. MANUAL (2001). Yeast host cells may then becultured using standard techniques known to those of ordinary skill inthe art.

Other methods for expressing heterologous proteins in yeast host cellsare well known to those of ordinary skill in the art. See generally U.S.Patent Publication No. 20020055169, U.S. Pat. Nos. 6,361,969; 6,312,923;6,183,985; 6,083,723; 6,017,731; 5,674,706; 5,629,203; 5,602,034; and5,089,398; U.S. Reexamined Patent Nos. RE37,343 and RE35,749; PCTPublished Patent Applications WO 99/07862; WO 98/37208; and WO 98/26080;European Patent Applications EP 0 946 736; EP 0 732 403; EP 0 480 480;EP 0 460 071; EP 0 340 986; EP 0 329 203; EP 0 324 274; and EP 0 164556. See also Gellissen et al., ANTONIE VAN LEEUWENHOEK (1992)62(1-2):79-93; Romanos et al., YEAST (1992) 8(6):423-488; Goeddel,METHODS IN ENZYMOLOGY (1990) 185:3-7, each incorporated by referenceherein.

The yeast host strains may be grown in fermentors during theamplification stage using standard feed batch fermentation methods wellknown to those of ordinary skill in the art. The fermentation methodsmay be adapted to account for differences in a particular yeast host'scarbon utilization pathway or mode of expression control. For example,fermentation of a Saccharomyces yeast host may require a single glucosefeed, complex nitrogen source (e.g., casein hydrolysates), and multiplevitamin supplementation. In contrast, the methylotrophic yeast P.pastoris may require glycerol, methanol, and trace mineral feeds, butonly simple ammonium (nitrogen) salts for optimal growth and expression.See, e.g., U.S. Pat. No. 5,324,639; Elliott et al., J. PROTEIN CHEM.(1990) 9:95; and Fieschko et al., BIOTECH. BIOENG. (1987) 29:1113,incorporated by reference herein.

Such fermentation methods, however, may have certain common featuresindependent of the yeast host strain employed. For example, a growthlimiting nutrient, typically carbon, may be added to the fermentorduring the amplification phase to allow maximal growth. In addition,fermentation methods generally employ a fermentation medium designed tocontain adequate amounts of carbon, nitrogen, basal salts, phosphorus,and other minor nutrients (vitamins, trace minerals and salts, etc.).Examples of fermentation media suitable for use with Pichia aredescribed in U.S. Pat. Nos. 5,324,639 and 5,231,178, which areincorporated by reference herein.

Baculovirus-Infected Insect Cells The term “insect host” or “insect hostcell” refers to a insect that can be, or has been, used as a recipientfor recombinant vectors or other transfer DNA. The term includes theprogeny of the original insect host cell that has been transfected. Itis understood that the progeny of a single parental cell may notnecessarily be completely identical in morphology or in genomic or totalDNA complement to the original parent, due to accidental or deliberatemutation. Progeny of the parental cell that are sufficiently similar tothe parent to be characterized by the relevant property, such as thepresence of a nucleotide sequence encoding a BPFI, are included in theprogeny intended by this definition.

The selection of suitable insect cells for expression of BPFI is wellknown to those of ordinary skill in the art. Several insect species arewell described in the art and are commercially available including Aedesaegypti, Bombyx mori, Drosophila melanogaster, Spodoptera frugiperda,and Trichoplusia ni. In selecting insect hosts for expression, suitablehosts may include those shown to have, inter alia, good secretioncapacity, low proteolytic activity, and overall robustness. Insect aregenerally available from a variety of sources including, but not limitedto, the Insect Genetic Stock Center, Department of Biophysics andMedical Physics, University of California (Berkeley, Calif.); and theAmerican Type Culture Collection (“ATCC”) (Manassas, Va.).

Generally, the components of a baculovirus-infected insect expressionsystem include a transfer vector, usually a bacterial plasmid, whichcontains both a fragment of the baculovirus genome, and a convenientrestriction site for insertion of the heterologous gene to be expressed;a wild type baculovirus with sequences homologous to thebaculovirus-specific fragment in the transfer vector (this allows forthe homologous recombination of the heterologous gene in to thebaculovirus genome); and appropriate insect host cells and growth media.The materials, methods and techniques used in constructing vectors,transfecting cells, picking plaques, growing cells in culture, and thelike are known in the art and manuals are available describing thesetechniques.

After inserting the heterologous gene into the transfer vector, thevector and the wild type viral genome are transfected into an insecthost cell where the vector and viral genome recombine. The packagedrecombinant virus is expressed and recombinant plaques are identifiedand purified. Materials and methods for baculovirus/insect cellexpression systems are commercially available in kit form from, forexample, Invitrogen Corp. (Carlsbad, Calif.). These techniques aregenerally known to those skilled in the art and fully described inSUMMERS AND SMITH, TEXAS AGRICULTURAL EXPERIMENT STATION BULLETIN NO.1555 (1987), herein incorporated by reference. See also, RICHARDSON, 39METHODS IN MOLECULAR BIOLOGY: BACULOVIRUS EXPRESSION PROTOCOLS (1995);AUSUBEL ET AL., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY 16.9-16.11(1994); KING AND P OSSEE, THE BACULOVIRUS SYSTEM: A LABORATORY GUIDE(1992); and O'REILLY ET AL., BACULOVIRUS EXPRESSION VECTORS: ALABORATORY MANUAL (1992).

Indeed, the production of various heterologous proteins usingbaculovirus/insect cell expression systems is well known in the art.See, e.g., U.S. Pat. Nos. 6,368,825; 6,342,216; 6,338,846; 6,261,805;6,245,528, 6,225,060; 6,183,987; 6,168,932; 6,126,944; 6,096,304;6,013,433; 5,965,393; 5,939,285; 5,891,676; 5,871,986; 5,861,279;5,858,368; 5,843,733; 5,762,939; 5,753,220; 5,605,827; 5,583,023;5,571,709; 5,516,657; 5,290,686; WO 02/06305; WO 01/90390; WO 01/27301;WO 01/05956; WO 00/55345; WO 00/20032 WO 99/51721; WO 99/45130; WO99/31257; WO 99/10515; WO 99/09193; WO 97/26332; WO 96/29400; WO96/25496; WO 96/06161; WO 95/20672; WO 93/03173; WO 92/16619; WO92/03628; WO 92/01801; WO 90/14428; WO 90/10078; WO 90/02566; WO90/02186; WO 90/01556; WO 89/01038; WO 89/01037; WO 88/07082, which areincorporated by reference herein.

Vectors that are useful in baculovirus/insect cell expression systemsare known in the art and include, for example, insect expression andtransfer vectors derived from the baculovirus Autographacalifornicanuclear polyhedrosis virus (AcNPV), which is a helper-independent, viralexpression vector. Viral expression vectors derived from this systemusually use the strong viral polyhedrin gene promoter to driveexpression of heterologous genes. See generally, O'Reilly ET AL.,BACULOVIRUS EXPRESSION VECTORS: A LABORATORY MANUAL (1992).

Prior to inserting the foreign gene into the baculovirus genome, theabove-described components, comprising a promoter, leader (if desired),coding sequence of interest, and transcription termination sequence, aretypically assembled into an intermediate transplacement construct(transfer vector). Intermediate transplacement constructs are oftenmaintained in a replicon, such as an extra chromosomal element (e.g.,plasmids) capable of stable maintenance in a host, such as bacteria. Thereplicon will have a replication system, thus allowing it to bemaintained in a suitable host for cloning and amplification. Morespecifically, the plasmid may contain the polyhedrin polyadenylationsignal (Miller, ANN. REV. MICROBIOL. (1988) 42:177) and a prokaryoticampicillin-resistance (amp) gene and origin of replication for selectionand propagation in E. coli.

One commonly used transfer vector for introducing foreign genes intoAcNPV is pAc373. Many other vectors, known to those of skill in the art,have also been designed including, for example, pVL985, which alters thepolyhedrin start codon from ATG to ATT, and which introduces a BamHIcloning site 32 base pairs downstream from the ATT. See Luckow andSummers, VIROLOGY 170:31 (1989). Other commercially available vectorsinclude, for example, PBlueBac4.5/V5-His; pBlueBacHis2; pMelBac;pBlueBac4.5 (Invitrogen Corp., Carlsbad, Calif.).

After insertion of the heterologous gene, the transfer vector and wildtype baculoviral genome are co-transfected into an insect cell host.Methods for introducing heterologous DNA into the desired site in thebaculovirus virus are known in the art. See SUMMERS AND SMITH, TEXASAGRICULTURAL EXPERIMENT STATION BULLETIN NO. 1555 (1987); Smith et al.,MOL. CELL. BIOL. (1983) 3:2156; Luckow and Summers, VIROLOGY (1989)170:31. For example, the insertion can be into a gene such as thepolyhedrin gene, by homologous double crossover recombination; insertioncan also be into a restriction enzyme site engineered into the desiredbaculovirus gene. See Miller et al., BIOESSAYS (1989) 11(4):91.

Transfection may be accomplished by electroporation. See TROTTER ANDWOOD, 39 METHODS IN MOLECULAR BIOLOGY (1995); Mann and King, J. GEN.VIROL. (1989) 70:3501. Alternatively, liposomes may be used to transfectthe insect cells with the recombinant expression vector and thebaculovirus. See, e.g., Liebman et al., BIOTECHNIQUES (1999) 26(1):36;Graves et al., BIOCHEMISTRY (1998) 37:6050; Nomura et al., J. BIOL.CHEM. (1998) 273(22):13570; Schmidt et al., PROTEIN EXPRESSION AND PURIFICATION (1998) 12:323; Siffert et al., NATURE GENETICS (1998) 18:45;TILKINS ET AL., CELL BIOLOGY: A LABORATORY HANDBOOK 145-154 (1998); Caiet al., PROTEIN EXPRESSION AND PURIFICATION (1997) 10:263; Dolphin etal., NATURE GENETICS (1997) 17:491; Kost et al., GENE (1997) 190:139;Jakobsson et al., J. BIOL. CHEM. (1996) 271:22203; Rowles et al., J.BIOL. CHEM. (1996) 271(37):22376; Reverey et al., J. BIOL. CHEM. (1996)271(39):23607-10; Stanley et al., J. BIOL. CHEM. (1995) 270:4121; Sisket al., J. VIROL. (1994) 68(2):766; and Peng et al., BIOTECHNIQUES(1993) 14(2):274. Commercially available liposomes include, for example,Cellfectin® and Lipofectin® (Invitrogen, Corp., Carlsbad, Calif.). Inaddition, calcium phosphate transfection may be used. See TROTTER ANDWOOD, 39 METHODS IN MOLECULAR BIOLOGY (1995); Kitts, NAR (1990)18(19):5667; and Mann and King, J. GEN VIROL. (1989) 70:3501.

Baculovirus expression vectors usually contain a baculovirus promoter. Abaculovirus promoter is any DNA sequence capable of binding abaculovirus RNA polymerase and initiating the downstream (3′)transcription of a coding sequence (e.g., structural gene) into mRNA. Apromoter will have a transcription initiation region which is usuallyplaced proximal to the 5′ end of the coding sequence. This transcriptioninitiation region typically includes an RNA polymerase binding site anda transcription initiation site. A baculovirus promoter may also have asecond domain called an enhancer, which, if present, is usually distalto the structural gene. Moreover, expression may be either regulated orconstitutive.

Structural genes, abundantly transcribed at late times in the infectioncycle, provide particularly useful promoter sequences. Examples includesequences derived from the gene encoding the viral polyhedron protein(FRIESEN ET AL., The Regulation of Baculovirus Gene Expression in THEMOLECULAR BIOLOGY OF BACULOVIRUSES (1986); EP 0 127 839 and 0 155 476)and the gene encoding the p10 protein (Vlak et al., J. GEN. VIROL.(1988) 69:765).

The newly formed baculovirus expression vector is packaged into aninfectious recombinant baculovirus and subsequently grown plaques may bepurified by techniques known to those skilled in the art. See Miller etal., BIOESSAYS (1989) 11(4):91; SUMMERS AND SMITH, TEXAS AGRICULTURALEXPERIMENT STATION BULLETIN N O. 1555 (1987).

Recombinant baculovirus expression vectors have been developed forinfection into several insect cells. For example, recombinantbaculoviruses have been developed for, inter alia, Aedes aegypti (ATCCNo. CCL-125), Bombyx mori (ATCC No. CRL-8910), Drosophila melanogaster(ATCC No. 1963), Spodoptera frugiperda, and Trichoplusia ni. See WO89/046,699; Wright, NATURE (1986) 321:718; Carbonell et al., J. VIROL.(1985) 56:153; Smith et al., MOL. CELL. BIOL. (1983) 3:2156. Seegenerally, Fraser et al., IN VITRO CELL. DEV. BIOL. (1989) 25:225. Morespecifically, the cell lines used for baculovirus expression vectorsystems commonly include, but are not limited to, Sf9 (Spodopterafrugiperda) (ATCC No. CRL-1711), Sf21 (Spodoptera frugiperda)(Invitrogen Corp., Cat. No. 11497-013 (Carlsbad, Calif.)), Tri-368(Trichopulsia ni), and High-Five™ BTI-TN-5B1-4 (Trichopulsia ni).

Cells and culture media are commercially available for both direct andfusion expression of heterologous polypeptides in abaculovirus/expression, and cell culture technology is generally knownto those skilled in the art.

E. Coli, Pseudomonas species, and other Prokaryotes Bacterial expressiontechniques are well known in the art. A wide variety of vectors areavailable for use in bacterial hosts. The vectors may be single copy orlow or high multicopy vectors. Vectors may serve for cloning and/orexpression. In view of the ample literature concerning vectors,commercial availability of many vectors, and even manuals describingvectors and their restriction maps and characteristics, no extensivediscussion is required here. As is well-known, the vectors normallyinvolve markers allowing for selection, which markers may provide forcytotoxic agent resistance, prototrophy or immunity. Frequently, aplurality of markers is present, which provide for differentcharacteristics.

A bacterial promoter is any DNA sequence capable of binding bacterialRNA polymerase and initiating the downstream (3′) transcription of acoding sequence (e.g. structural gene) into mRNA. A promoter will have atranscription initiation region which is usually placed proximal to the5′ end of the coding sequence. This transcription initiation regiontypically includes an RNA polymerase binding site and a transcriptioninitiation site. A bacterial promoter may also have a second domaincalled an operator, that may overlap an adjacent RNA polymerase bindingsite at which RNA synthesis begins. The operator permits negativeregulated (inducible) transcription, as a gene repressor protein maybind the operator and thereby inhibit transcription of a specific gene.Constitutive expression may occur in the absence of negative regulatoryelements, such as the operator. In addition, positive regulation may beachieved by a gene activator protein binding sequence, which, if presentis usually proximal (5′) to the RNA polymerase binding sequence. Anexample of a gene activator protein is the catabolite activator protein(CAP), which helps initiate transcription of the lac operon inEscherichia coli (E. coli) [Raibaud et al., ANNU. REV. GENET. (1984)18:173]. Regulated expression may therefore be either positive ornegative, thereby either enhancing or reducing transcription.

Sequences encoding metabolic pathway enzymes provide particularly usefulpromoter sequences. Examples include promoter sequences derived fromsugar metabolizing enzymes, such as galactose, lactose (lac) [Chang etal., NATURE (1977) 198:1056], and maltose. Additional examples includepromoter sequences derived from biosynthetic enzymes such as tryptophan(trp) [Goeddel et al., NUC. ACIDS RES. (1980) 8:4057; Yelverton et al.,NUCL. ACIDS RES. (1981) 9:731; U.S. Pat. No. 4,738,921; EP Pub. Nos. 036776 and 121 775, which are incorporated by reference herein]. Theβ-galactosidase (bla) promoter system [Weissmann (1981) “The cloning ofinterferon and other mistakes.” In Interferon 3 (Ed. I. Gresser)],bacteriophage lambda PL [Shimatake et al. NATURE (1981) 292:128] and T5[U.S. Pat. No. 4,689,406, which are incorporated by reference herein]promoter systems also provide useful promoter sequences. Preferredmethods of the present invention utilize strong promoters, such as theT7 promoter to induce BPFI at high levels. Examples of such vectors arewell known in the art and include the pET29 series from Novagen, and thepPOP vectors described in WO99/05297, which is incorporated by referenceherein. Such expression systems produce high levels of BPFI in the hostwithout compromising host cell viability or growth parameters.

In addition, synthetic promoters which do not occur in nature alsofunction as bacterial promoters. For example, transcription activationsequences of one bacterial or bacteriophage promoter may be joined withthe operon sequences of another bacterial or bacteriophage promoter,creating a synthetic hybrid promoter [U.S. Pat. No. 4,551,433, which isincorporated by reference herein]. For example, the tac promoter is ahybrid trp-lac promoter comprised of both trp promoter and lac operonsequences that is regulated by the lac repressor [Amann et al., GENE(1983) 25:167; de Boer et al., PROC. NATL. ACAD. SCI. (1983) 80:21].Furthermore, a bacterial promoter can include naturally occurringpromoters of non-bacterial origin that have the ability to bindbacterial RNA polymerase and initiate transcription. A naturallyoccurring promoter of non-bacterial origin can also be coupled with acompatible RNA polymerase to produce high levels of expression of somegenes in prokaryotes. The bacteriophage T7 RNA polymerase/promotersystem is an example of a coupled promoter system [Studier et al., J.MOL. BIOL. (1986) 189:113; Tabor et al., Proc Natl. Acad. Sci. (1985)82:1074]. In addition, a hybrid promoter can also be comprised of abacteriophage promoter and an E. coli operator region (EP Pub. No. 267851).

In addition to a functioning promoter sequence, an efficient ribosomebinding site is also useful for the expression of foreign genes inprokaryotes. In E. coli, the ribosome binding site is called theShine-Dalgarno (SD) sequence and includes an initiation codon (ATG) anda sequence 3-9 nucleotides in length located 3-11 nucleotides upstreamof the initiation codon [Shine et al., NATURE (1975) 254:34]. The SDsequence is thought to promote binding of mRNA to the ribosome by thepairing of bases between the SD sequence and the 3′ and of E. coli 16SrRNA [Steitz et al. “Genetic signals and nucleotide sequences inmessenger RNA”, In Biological Regulation and Development: GeneExpression (Ed. R. F. Goldberger, 1979)]. To express eukaryotic genesand prokaryotic genes with weak ribosome-binding site [Sambrook et al.“Expression of cloned genes in Escherichia coli”, Molecular Cloning: ALaboratory Manual, 1989]

The term “bacterial host” or “bacterial host cell” refers to a bacterialthat can be, or has been, used as a recipient for recombinant vectors orother transfer DNA. The term includes the progeny of the originalbacterial host cell that has been transfected. It is understood that theprogeny of a single parental cell may not necessarily be completelyidentical in morphology or in genomic or total DNA complement to theoriginal parent, due to accidental or deliberate mutation. Progeny ofthe parental cell that are sufficiently similar to the parent to becharacterized by the relevant property, such as the presence of anucleotide sequence encoding a BPFI, are included in the progenyintended by this definition.

The selection of suitable host bacteria for expression of BPFI is wellknown to those of ordinary skill in the art. In selecting bacterialhosts for expression, suitable hosts may include those shown to have,inter alia, good inclusion body formation capacity, low proteolyticactivity, and overall robustness. Bacterial hosts are generallyavailable from a variety of sources including, but not limited to, theBacterial Genetic Stock Center, Department of Biophysics and MedicalPhysics, University of California (Berkeley, Calif.); and the AmericanType Culture Collection (“ATCC”) (Manassas, Va.).Industrial/pharmaceutical fermentation generally use bacterial derivedfrom K strains (e.g. W3110) or from bacteria derived from B strains(e.g. BL21). These strains are particularly useful because their growthparameters are extremely well known and robust. In addition, thesestrains are non-pathogenic, which is commercially important for safetyand environmental reasons. In one embodiment of the methods of thepresent invention, the E. coli host is a strain of BL21. In anotherembodiment of the methods of the present invention, the E. coli host isa protease minus strain including, but not limited to, OMP- and LON-. Inanother embodiment of the methods of the present invention, the hostcell strain is a species of Pseudomonas, including but not limited to,Pseudomonas fluorescens, Pseudomonas aeruginosa, and Pseudomonas putida.Pseudomonas fluorescens biovar 1, designated strain MB101, is known tobe useful for recombinant production and is available for therapeuticprotein production processes. Examples of a Pseudomonas expressionsystem include the system available by The Dow Chemical Company as ahost strain (Midland, Mich. available on the World Wide Web at dow.com).U.S. Pat. Nos. 4,755,465 and 4,859,600, which are incorporated byreference herein, describe the use of Pseudomonas strains as a host cellfor hGH production.

Once a recombinant host cell strain has been established (i.e., theexpression construct has been introduced into the host cell and hostcells with the proper expression construct are isolated), therecombinant host cell strain is cultured under conditions appropriatefor production of BPFI. As will be apparent to one of skill in the art,the method of culture of the recombinant host cell strain will bedependent on the nature of the expression construct utilized and theidentity of the host cell. Recombinant host strains are normallycultured using methods that are well known to the art. Recombinant hostcells are typically cultured in liquid medium containing assimilatablesources of carbon, nitrogen, and inorganic salts and, optionally,containing vitamins, amino acids, growth factors, and otherproteinaceous culture supplements well known to the art. Liquid mediafor culture of host cells may optionally contain antibiotics oranti-fungals to prevent the growth of undesirable microorganisms and/orcompounds including, but not limited to, antibiotics to select for hostcells containing the expression vector.

Recombinant host cells may be cultured in batch or continuous formats,with either cell harvesting (in the case where BPFI accumulatesintracellularly) or harvesting of culture supernatant in either batch orcontinuous formats. For production in prokaryotic host cells, batchculture and cell harvest are preferred.

The BPFIs of the present invention are normally purified afterexpression in recombinant systems. The BPFI may be purified from hostcells by a variety of methods known to the art. Normally, BPFI producedin bacterial host cells may be poorly soluble or insoluble (in the formof inclusion bodies). In one embodiment of the present invention, aminoacid substitutions may readily be made in the BPFI that are selected forthe purpose of increasing the solubility of the recombinantly producedprotein utilizing the methods disclosed herein as well as those known inthe art. In the case of insoluble protein, the protein may be collectedfrom host cell lysates by centrifugation and may further be followed byhomogenization of the cells. In the case of poorly soluble protein,compounds including, but not limited to, polyethylene imine (PEI) may beadded to induce the precipitation of partially soluble protein. Theprecipitated protein may then be conveniently collected bycentrifugation. Recombinant host cells may be disrupted or homogenizedto release the inclusion bodies from within the cells using a variety ofmethods well known to those of ordinary skill in the art. Host celldisruption or homogenization may be performed using well knowntechniques including, but not limited to, enzymatic cell disruption,sonication, dounce homogenization, or high pressure release disruption.In one embodiment of the method of the present invention, the highpressure release technique may be used to disrupt the E. coli host cellsto release the inclusion bodies of BPFI. When handling inclusion bodiesof BPFI, it may be advantageous to minimize the homogenization time onrepetitions in order to maximize the yield of inclusion bodies withoutloss due to factors such as solubilization, mechanical shearing orproteolysis. The tendency for the formation of inclusion bodies may beenhanced by fusion of the target protein to certain other proteins, suchas TrpLE [Georgiou, G. (1996) in Protein engineering: Principles andPractice (Cleland, J. L. and Craik, C. S., eds.), pp. 101-127,Wiley-Liss, New York, Ford, C. F., Suominen, I. and Glatz, C. E. (1991)Protein Expression Purif. 2, 95-107], and by cultivation at elevatedtemperatures or at a pH other than 7.0.

Insoluble or precipitated BPFI may then be solubilized using any of anumber of suitable solubilization agents known to the art. Preferably,BPFI is solubilized with urea or guanidine hydrochloride. The volume ofthe solubilized BPFI should be minimized so that large batches may beproduced using conveniently manageable batch sizes. This factor may besignificant in a large-scale commercial setting where the recombinanthost may be grown in batches that are thousands of liters in volume. Inaddition, when manufacturing BPFI in a large-scale commercial setting,in particular for human pharmaceutical uses, the avoidance of harshchemicals that can damage the machinery and container, or the proteinproduct itself, should be avoided, if possible. It has been shown in themethod of the present invention that the milder denaturing agent ureacan be used to solubilize the BPFI inclusion bodies in place of theharsher denaturing agent guanidine hydrochloride. The use of ureasignificantly reduces the risk of damage to stainless steel equipmentutilized in the manufacturing and purification process of BPFI whileefficiently solubilizing the BPFI inclusion bodies.

In the case of soluble BPFI, the BPFI may be secreted into theperiplasmic space or into the culture medium. In addition, soluble BPFImay be present in the cytoplasm of the host cells. It may be desired toconcentrate soluble BPFI prior to performing purification steps.Standard techniques known to those skilled in the art may be used toconcentrate soluble BPFI from, for example, cell lysates or culturemedium. In addition, standard techniques known to those skilled in theart may be used to disrupt host cells and release soluble BPFI from thecytoplasm or periplasmic space of the host cells.

When BPFI is produced as a fusion protein, the fusion sequence ispreferably removed. Removal of a fusion sequence may be accomplishedunder a number of different conditions, including but not limited to, byenzymatic or chemical cleavage. Enzymatic removal of fusion sequencesmay be accomplished using methods well known to those in the art. Thechoice of enzyme for removal of the fusion sequence will be determinedby the identity of the fusion, and the reaction conditions will bespecified by the choice of enzyme as will be apparent to one skilled inthe art. Chemical cleavage may be accomplished using reagents well knownto those in the art. One such reagent is cyanogen bromide which cleavesat methionine residues. The cleaved BPFI is preferably purified from thecleaved fusion sequence by well known methods. Such methods will bedetermined by the identity and properties of the fusion sequence andBPFI, as will be apparent to one skilled in the art. Peptide bonds forremoval of fusion sequence, for example, may be cleaved under exposureto photon energy, increased temperature, decreased temperature,increased pH, decreased pH, exposure to sub-atomic particles, additionof a catalyst, incubation with an enzyme, contact with another chemicalfunctional group, and/or other conditions. For a peptide bond to becleaved under one or more of these conditions, the non-naturally encodedamino acid may have a functional group with one or more characteristicsincluding, but not limited to, a photo-activated functional group, pHactivated functional group, temperature activated functional group,functional group that requires a catalyst, and a functional group thatis recognized by a protease, enzyme, or another chemical functionalgroup. Methods for purification may include, but are not limited to,size-exclusion chromatography, hydrophobic interaction chromatography,ion-exchange chromatography or dialysis or any combination thereof.

The BPFI is also preferably purified to remove DNA from the proteinsolution. DNA may be removed by any suitable method known to the art,such as precipitation or ion exchange chromatography, but is preferablyremoved by precipitation with a nucleic acid precipitating agent, suchas, but not limited to, protamine sulfate. BPFI may be separated fromthe precipitated DNA using standard well known methods including, butnot limited to, centrifugation or filtration. Removal of host nucleicacid molecules is an important factor in a setting where BPFI is to beused to treat humans and the methods of the present invention reducehost cell DNA to pharmaceutically acceptable levels.

Methods for small-scale or large-scale fermentation can also be used inprotein expression, including but not limited to, fermentors, shakeflasks, fluidized bed bioreactors, hollow fiber bioreactors, rollerbottle culture systems, and stirred tank bioreactor systems. Each ofthese methods can be performed in a batch, fed-batch, or continuous modeprocess.

Human BPFIs of the invention can generally be recovered using methodsstandard in the art. For example, culture medium or cell lysate can becentrifuged or filtered to remove cellular debris. The supernatant maybe concentrated or diluted to a desired volume or diafiltered into asuitable buffer to condition the preparation for further purification.Further purification of the BPFI of the present invention includesseparating deamidated and clipped forms of the BPFI variant from theintact form.

Any of the following exemplary procedures can be employed forpurification of BPFIs of the invention: affinity chromatography; anion-or cation-exchange chromatography (using, including but not limited to,DEAE SEPHAROSE); chromatography on silica; reverse phase HPLC; gelfiltration (using, including but not limited to, SEPHADEX G-75);hydrophobic interaction chromatography; size-exclusion chromatography,metal-chelate chromatography; ultrafiltration/diafiltration; ethanolprecipitation; ammonium sulfate precipitation; chromatofocusing;displacement chromatography; electrophoretic procedures (including butnot limited to preparative isoelectric focusing), differentialsolubility (including but not limited to ammonium sulfateprecipitation), SDS-PAGE, or extraction.

Proteins of the present invention, including but not limited to,proteins comprising unnatural amino acids, peptides comprising unnaturalamino acids, antibodies to proteins comprising unnatural amino acids,binding partners for proteins comprising unnatural amino acids, etc.,can be purified, either partially or substantially to homogeneity,according to standard procedures known to and used by those of skill inthe art. Accordingly, polypeptides of the invention can be recovered andpurified by any of a number of methods well known in the art, includingbut not limited to, ammonium sulfate or ethanol precipitation, acid orbase extraction, column chromatography, affinity column chromatography,anion or cation exchange chromatography, phosphocellulosechromatography, hydrophobic interaction chromatography, hydroxylapatitechromatography, lectin chromatography, gel electrophoresis and the like.Protein refolding steps can be used, as desired, in making correctlyfolded mature proteins. High performance liquid chromatography (HPLC),affinity chromatography or other suitable methods can be employed infinal purification steps where high purity is desired. In oneembodiment, antibodies made against unnatural amino acids (or proteinsor peptides comprising unnatural amino acids) are used as purificationreagents, including but not limited to, for affinity-based purificationof proteins or peptides comprising one or more unnatural amino acid(s).Once purified, partially or to homogeneity, as desired, the polypeptidesare optionally used for a wide variety of utilities, including but notlimited to, as assay components, therapeutics, prophylaxis, diagnostics,research reagents, and/or as immunogens for antibody production.

In addition to other references noted herein, a variety ofpurification/protein folding methods are well known in the art,including, but not limited to, those set forth in R. Scopes, ProteinPurification, Springer-Verlag, N.Y. (1982); Deutscher, Methods inEnzymology Vol. 182: Guide to Protein Purification, Academic Press, Inc.N.Y. (1990); Sandana, (1997) Bioseparation of Proteins, Academic Press,Inc.; Bollag et al. (1996) Protein Methods, 2nd Edition Wiley-Liss, NY;Walker, (1996) The Protein Protocols Handbook Humana Press, NJ, Harrisand Angal, (1990) Protein Purification Applications: A PracticalApproach IRL Press at Oxford, Oxford, England; Harris and Angal, ProteinPurification Methods: A Practical Approach IRL Press at Oxford, Oxford,England; Scopes, (1993) Protein Purification: Principles and Practice3rd Edition Springer Verlag, NY; Janson and Ryden, (1998) ProteinPurification: Principles, High Resolution Methods and Applications,Second Edition Wiley-VCH, NY; and Walker (1998), Protein Protocols onCD-ROM Humana Press, NJ; and the references cited therein.

One advantage of producing a protein or polypeptide of interest with anunnatural amino acid in a eukaryotic host cell or non-eukaryotic hostcell is that typically the proteins or polypeptides will be folded intheir native conformations. However, in certain embodiments of theinvention, those of skill in the art will recognize that, aftersynthesis, expression and/or purification, proteins, or peptides canpossess a conformation different from the desired conformations of therelevant polypeptides. In one aspect of the invention, the expressedprotein or polypeptide is optionally denatured and then renatured. Thisis accomplished utilizing methods known in the art, including but notlimited to, by adding a chaperonin to the protein or polypeptide ofinterest, by solubilizing the proteins in a chaotropic agent such asguanidine HCl, utilizing protein disulfide isomerase, etc.

In general, it is occasionally desirable to denature and reduceexpressed polypeptides and then to cause the polypeptides to re-foldinto the preferred conformation. For example, guanidine, urea, DTT, DTE,and/or a chaperonin can be added to a translation product of interest.Methods of reducing, denaturing and renaturing proteins are well knownto those of skill in the art (see, the references above, and Debinski,et al. (1993) J. Biol. Chem., 268: 14065-14070; Kreitman and Pastan(1993) Bioconjug. Chem., 4: 581-585; and Buchner, et al., (1992) Anal.Biochem., 205: 263-270). Debinski, et al., for example, describe thedenaturation and reduction of inclusion body proteins in guanidine-DTE.The proteins can be refolded in a redox buffer containing, including butnot limited to, oxidized glutathione and L-arginine. Refolding reagentscan be flowed or otherwise moved into contact with the one or morepolypeptide or other expression product, or vice-versa.

In the case of prokaryotic production of BPFI, the BPFI thus producedmay be misfolded and thus lacks or has reduced biological activity. Thebioactivity of the protein may be restored by “refolding”. In general,misfolded BPFI is refolded by solubilizing (where the BPFI is alsoinsoluble), unfolding and reducing the polypeptide chain using, forexample, one or more chaotropic agents (e.g. urea and/or guanidine) anda reducing agent capable of reducing disulfide bonds (e.g.dithiothreitol, DTT or 2-mercaptoethanol, 2-ME). At a moderateconcentration of chaotrope, an oxidizing agent is then added (e.g.,oxygen, cystine or cystamine), which allows the reformation of disulfidebonds. BPFI may be refolded using standard methods known in the art,such as those described in U.S. Pat. Nos. 4,511,502, 4,511,503, and4,512,922, which are incorporated by reference herein. The BPFI may alsobe cofolded with other proteins to form heterodimers or heteromultimers.After refolding or cofolding, the BPFI is preferably further purified.

General Purification Methods Any one of a variety of isolation steps maybe performed on the cell lysate comprising BPFI or on any BPFI mixturesresulting from any isolation steps including, but not limited to,affinity chromatography, ion exchange chromatography, hydrophobicinteraction chromatography, gel filtration chromatography, highperformance liquid chromatography (“HPLC”), reversed phase-HPLC(“RP-HPLC”), expanded bed adsorption, or any combination and/orrepetition thereof and in any appropriate order.

Equipment and other necessary materials used in performing thetechniques described herein are commercially available. Pumps, fractioncollectors, monitors, recorders, and entire systems are available from,for example, Applied Biosystems (Foster City, Calif.), Bio-RadLaboratories, Inc. (Hercules, Calif.), and Amersham Biosciences, Inc.(Piscataway, N.J.). Chromatographic materials including, but not limitedto, exchange matrix materials, media, and buffers are also availablefrom such companies.

Equilibration, and other steps in the column chromatography processesdescribed herein such as washing and elution, may be more rapidlyaccomplished using specialized equipment such as a pump. Commerciallyavailable pumps include, but are not limited to, HILOAD® Pump P-50,Peristaltic Pump P-1, Pump P-901, and Pump P-903 (Amersham Biosciences,Piscataway, N.J.).

Examples of fraction collectors include RediFrac Fraction Collector,FRAC-100 and FRAC-200 Fraction Collectors, and SUPERFRAC® FractionCollector (Amersham Biosciences, Piscataway, N.J.). Mixers are alsoavailable to form pH and linear concentration gradients. Commerciallyavailable mixers include Gradient Mixer GM-1 and In-Line Mixers(Amersham Biosciences, Piscataway, N.J.).

The chromatographic process may be monitored using any commerciallyavailable monitor. Such monitors may be used to gather information likeUV, pH, and conductivity. Examples of detectors include Monitor UV-1,UVICORD® S II, Monitor UV-M II, Monitor UV-900, Monitor UPC-900, MonitorpH/C-900, and Conductivity Monitor (Amersham Biosciences, Piscataway,N.J.). Indeed, entire systems are commercially available including thevarious AKTA® systems from Amersham Biosciences (Piscataway, N.J.).

In one embodiment of the present invention, for example, the BPFI may bereduced and denatured by first denaturing the resultant purified BPFI inurea, followed by dilution into TRIS buffer containing a reducing agent(such as DTT) at a suitable pH. In another embodiment, the BPFI isdenatured in urea in a concentration range of between about 2 M to about9 M, followed by dilution in TRIS buffer at a pH in the range of about5.0 to about 8.0. The refolding mixture of this embodiment may then beincubated. In one embodiment, the refolding mixture is incubated at roomtemperature for four to twenty-four hours. The reduced and denaturedBPFI mixture may then be further isolated or purified.

As stated herein, the pH of the first BPFI mixture may be adjusted priorto performing any subsequent isolation steps. In addition, the firstBPFI mixture or any subsequent mixture thereof may be concentrated usingtechniques known in the art. Moreover, the elution buffer comprising thefirst BPFI mixture or any subsequent mixture thereof may be exchangedfor a buffer suitable for the next isolation step using techniques wellknown to those of ordinary skill in the art.

Ion Exchange Chromatography In one embodiment, and as an optional,additional step, ion exchange chromatography may be performed on thefirst BPFI mixture. See generally ION EXCHANGE CHROMATOGRAPHY:PRINCIPLES AND METHODS (Cat. No. 18-1114-21, Amersham Biosciences(Piscataway, N.J.)). Commercially available ion exchange columns includeHITRAP®, HIPREP®, and HILOAD® Columns (Amersham Biosciences, Piscataway,N.J.). Such columns utilize strong anion exchangers such as Q SEPHAROSE®Fast Flow, Q SEPHAROSE® High Performance, and Q SEPHAROSE® XL; strongcation exchangers such as SP SEPHAROSE® High Performance, SP SEPHAROSE®Fast Flow, and SP SEPHAROSE® XL; weak anion exchangers such as DEAESEPHAROSE® Fast Flow; and weak cation exchangers such as CM SEPHAROSE®Fast Flow (Amersham Biosciences, Piscataway, N.J.). Anion or cationexchange column chromatography may be performed on the BPFI at any stageof the purification process to isolate substantially purified BPFI. Thecation exchange chromatography step may be performed using any suitablecation exchange matrix. Useful cation exchange matrices include, but arenot limited to, fibrous, porous, non-porous, microgranular, beaded, orcross-linked cation exchange matrix materials. Such cation exchangematrix materials include, but are not limited to, cellulose, agarose,dextran, polyacrylate, polyvinyl, polystyrene, silica, polyether, orcomposites of any of the foregoing.

The cation exchange matrix may be any suitable cation exchangerincluding strong and weak cation exchangers. Strong cation exchangersmay remain ionized over a wide pH range and thus, may be capable ofbinding BPFI over a wide pH range. Weak cation exchangers, however, maylose ionization as a function of pH. For example, a weak cationexchanger may lose charge when the pH drops below about pH 4 or pH 5.Suitable strong cation exchangers include, but are not limited to,charged functional groups such as sulfopropyl (SP), methyl sulfonate(S), or sulfoethyl (SE). The cation exchange matrix may be a strongcation exchanger, preferably having a BPFI binding pH range of about 2.5to about 6.0. Alternatively, the strong cation exchanger may have a BPFIbinding pH range of about pH 2.5 to about pH 5.5. The cation exchangematrix may be a strong cation exchanger having a BPFI binding pH ofabout 3.0. Alternatively, the cation exchange matrix may be a strongcation exchanger, preferably having a BPFI binding pH range of about 6.0to about 8.0. The cation exchange matrix may be a strong cationexchanger preferably having a BPFI binding pH range of about 8.0 toabout 12.5. Alternatively, the strong cation exchanger may have a BPFIbinding pH range of about pH 8.0 to about pH 12.0.

Prior to loading the BPFI, the cation exchange matrix may beequilibrated, for example, using several column volumes of a dilute,weak acid, e.g., four column volumes of 20 mM acetic acid, pH 3.Following equilibration, the BPFI may be added and the column may bewashed one to several times, prior to elution of substantially purifiedBPFI, also using a weak acid solution such as a weak acetic acid orphosphoric acid solution. For example, approximately 2-4 column volumesof 20 mM acetic acid, pH 3, may be used to wash the column. Additionalwashes using, e.g., 2-4 column volumes of 0.05 M sodium acetate, pH 5.5,or 0.05 M sodium acetate mixed with 0.1 M sodium chloride, pH 5.5, mayalso be used. Alternatively, using methods known in the art, the cationexchange matrix may be equilibrated using several column volumes of adilute, weak base.

Alternatively, substantially purified BPFI may be eluted by contactingthe cation exchanger matrix with a buffer having a sufficiently low pHor ionic strength to displace the BPFI from the matrix. The pH of theelution buffer may range from about pH 2.5 to about pH 6.0. Morespecifically, the pH of the elution buffer may range from about pH 2.5to about pH 5.5, about pH 2.5 to about pH 5.0. The elution buffer mayhave a pH of about 3.0. In addition, the quantity of elution buffer mayvary widely and will generally be in the range of about 2 to about 10column volumes.

Following adsorption of BPFI to the cation exchanger matrix,substantially purified BPFI may be eluted by contacting the matrix witha buffer having a sufficiently high pH or ionic strength to displaceBPFI from the matrix. Suitable buffers for use in high pH elution ofsubstantially purified BPFI include, but are not limited to, citrate,phosphate, formate, acetate, HEPES, and MES buffers ranging inconcentration from at least about 5 mM to at least about 100 mM.

Reverse-Phase Chromatography RP-HPLC may be performed to purify proteinsfollowing suitable protocols that are known to those of ordinary skillin the art. See, e.g., Pearson et al., ANAL BIOCHEM. (1982) 124:217-230(1982); Rivier et al., J. CHROM. (1983) 268:112-119; Kunitani et al., J.CHROM. (1986) 359:391-402. RP-HPLC may be performed on the BPFI toisolate substantially purified BPFI. In this regard, silica derivatizedresins with alkyl functionalities with a wide variety of lengths,including, but not limited to, at least about C₃ to at least about C₃₀,at least about C₃ to at least about C₂₀, or at least about C₃ to atleast about C₁₈, resins may be used. Alternatively, a polymeric resinmay be used. For example, TosoHaas Amberchrome CG1000sd resin may beused, which is a styrene polymer resin. Cyano or polymeric resins with awide variety of alkyl chain lengths may also be used. Furthermore, theRP-HPLC column may be washed with a solvent such as ethanol. The SourceRP column is another example of a RP-HPLC column.

A suitable elution buffer containing an ion pairing agent and an organicmodifier such as methanol, isopropanol, tetrahydrofuran, acetonitrile orethanol, may be used to elute the BPFI from the RP-HPLC column. The mostcommonly used ion pairing agents include, but are not limited to, aceticacid, formic acid, perchloric acid, phosphoric acid, trifluoroaceticacid, heptafluorobutyric acid, triethylamine, tetramethylammonium,tetrabutylammonium, triethylammonium acetate. Elution may be performedusing one or more gradients or isocratic conditions, with gradientconditions preferred to reduce the separation time and to decrease peakwidth. Another method involves the use of two gradients with differentsolvent concentration ranges. Examples of suitable elution buffers foruse herein may include, but are not limited to, ammonium acetate andacetonitrile solutions.

Hydrophobic Interaction Chromatography Purification TechniquesHydrophobic interaction chromatography (HIC) may be performed on theBPFI. See generally HYDROPHOBIC INTERACTION CHROMATOGRAPHY HANDBOOK:PRINCIPLES AND METHODS (Cat. No. 18-1020-90, Amersham Biosciences(Piscataway, N.J.) which is incorporated by reference herein. SuitableHIC matrices may include, but are not limited to, alkyl- oraryl-substituted matrices, such as butyl-, hexyl-, octyl- orphenyl-substituted matrices including agarose, cross-linked agarose,sepharose, cellulose, silica, dextran, polystyrene, poly(methacrylate)matrices, and mixed mode resins, including but not limited to, apolyethyleneamine resin or a butyl- or phenyl-substitutedpoly(methacrylate) matrix. Commercially available sources forhydrophobic interaction column chromatography include, but are notlimited to, HITRAP®, HIPREP®, and HILOAD® columns (Amersham Biosciences,Piscataway, N.J.).

Briefly, prior to loading, the HIC column may be equilibrated usingstandard buffers known to those of ordinary skill in the art, such as anacetic acid/sodium chloride solution or HEPES containing ammoniumsulfate. After loading the BPFI, the column may then washed usingstandard buffers and conditions to remove unwanted materials butretaining the BPFI on the HIC column. BPFI may be eluted with about 3 toabout 10 column volumes of a standard buffer, such as a HEPES buffercontaining EDTA and lower ammonium sulfate concentration than theequilibrating buffer, or an acetic acid/sodium chloride buffer, amongothers. A decreasing linear salt gradient using, for example, a gradientof potassium phosphate, may also be used to elute the BPFI molecules.The eluant may then be concentrated, for example, by filtration such asdiafiltration or ultrafiltration. Diafiltration may be utilized toremove the salt used to elute BPFI.

Other Purification Techniques Yet another isolation step using, forexample, gel filtration (GEL FILTRATION: PRINCIPLES AND METHODS (Cat.No. 18-1022-18, Amersham Biosciences, Piscataway, N.J.) which isincorporated by reference herein, hydroxyapatite chromatography(suitable matrices include, but are not limited to, HA-Ultrogel, HighResolution (Calbiochem), CHT Ceramic Hydroxyapatite (BioRad), Bio-GelHTP Hydroxyapatite (BioRad)), HPLC, expanded bed adsorption,ultrafiltration, diafiltration, lyophilization, and the like, may beperformed on the first BPFI mixture or any subsequent mixture thereof,to remove any excess salts and to replace the buffer with a suitablebuffer for the next isolation step or even formulation of the final drugproduct.

The yield of BPFI, including substantially purified BPFI, may bemonitored at each step described herein using techniques known to thoseof ordinary skill in the art. Such techniques may also used to assessthe yield of substantially purified BPFI following the last isolationstep. For example, the yield of BPFI may be monitored using any ofseveral reverse phase high pressure liquid chromatography columns,having a variety of alkyl chain lengths such as cyano RP-HPLC,C₁₈RP-HPLC; as well as cation exchange HPLC and gel filtration HPLC.

In specific embodiments of the present invention, the yield of BPFIafter each purification step may be at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 55%, at least about 60%, at least about 65%, at least about70%, at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 91%, at least about 92%, at least about93%, at least about 94%, at least about 95%, at least about 96%, atleast about 97%, at least about 98%, at least about 99%, at least about99.9%, or at least about 99.99%, of the BPFI in the starting materialfor each purification step.

Purity may be determined using standard techniques, such as SDS-PAGE, orby measuring BPFI using Western blot and ELISA assays. For example,polyclonal antibodies may be generated against proteins isolated fromnegative control yeast fermentation and the cation exchange recovery.The antibodies may also be used to probe for the presence ofcontaminating host cell proteins.

RP-HPLC material Vydac C4 (Vydac) consists of silica gel particles, thesurfaces of which carry C4-alkyl chains. The separation of BPFI from theproteinaceous impurities is based on differences in the strength ofhydrophobic interactions. Elution is performed with an acetonitrilegradient in diluted trifluoroacetic acid. Preparative HPLC is performedusing a stainless steel column (filled with 2.8 to 3.2 liter of Vydac C4silicagel). The Hydroxyapatite Ultrogel eluate is acidified by addingtrifluoroacetic acid and loaded onto the Vydac C4 column. For washingand elution an acetonitrile gradient in diluted trifluoroacetic acid isused. Fractions are collected and immediately neutralized with phosphatebuffer. The BPFI fractions which are within the IPC limits are pooled.

DEAE Sepharose (Pharmacia) material consists of diethylaminoethyl(DEAE)-groups which are covalently bound to the surface of Sepharosebeads. The binding of BPFI to the DEAE groups is mediated by ionicinteractions. Acetonitrile and trifluoroacetic acid pass through thecolumn without being retained. After these substances have been washedoff, trace impurities are removed by washing the column with acetatebuffer at a low pH. Then the column is washed with neutral phosphatebuffer and BPFI is eluted with a buffer with increased ionic strength.The column is packed with DEAE Sepharose fast flow. The column volume isadjusted to assure a BPFI load in the range of 3-10 mg BPFI/ml gel. Thecolumn is washed with water and equilibration buffer (sodium/potassiumphosphate). The pooled fractions of the HPLC eluate are loaded and thecolumn is washed with equilibration buffer. Then the column is washedwith washing buffer (sodium acetate buffer) followed by washing withequilibration buffer. Subsequently, BPFI is eluted from the column withelution buffer (sodium chloride, sodium/potassium phosphate) andcollected in a single fraction in accordance with the master elutionprofile. The eluate of the DEAE Sepharose column is adjusted to thespecified conductivity. The resulting drug substance is sterile filteredinto Teflon bottles and stored at −70° C.

Additional methods that may be employed include, but are not limited to,steps to remove endotoxins. Endotoxins are lipopoly-saccharides (LPSs)which are located on the outer membrane of Gram-negative host cells,such as, for example, Escherichia coli. Methods for reducing endotoxinlevels are known to one skilled in the art and include, but are notlimited to, purification techniques using silica supports, glass powderor hydroxyapatite, reverse-phase, affinity, size-exclusion,anion-exchange chromatography, hydrophobic interaction chromatography, acombination of these methods, and the like. Modifications or additionalmethods may be required to remove contaminants such as co-migratingproteins from the polypeptide of interest.

A wide variety of methods and procedures can be used to assess the yieldand purity of a BPFI comprising one or more non-naturally encoded aminoacids, including but not limited to, the Bradford assay, SDS-PAGE,silver stained SDS-PAGE, coomassie stained SDS-PAGE, mass spectrometry(including but not limited to, MALDI-TOF) and other methods forcharacterizing proteins known to one skilled in the art.

Characterization of the Heterologous Fusion Proteins of the PresentInvention

Numerous methods exist to characterize the fusion proteins of thepresent invention. Some of these methods include, but are not limitedto: SDS-PAGE coupled with protein staining methods or immunoblottingusing anti-IgG or anti-HSA antibodies. Other methods include matrixassisted laser desorption/ionization-mass spectrometry (MALDI-MS),liquid chromatography/mass spectrometry, isoelectric focusing,analytical anion exchange, chromatofocusing, and circular dichroism, forexample.

VIII. Expression in Alternate Systems

Several strategies have been employed to introduce unnatural amino acidsinto proteins in non-recombinant host cells, mutagenized host cells, orin cell-free systems. These systems are also suitable for use in makingthe BPFIs of the present invention. Derivatization of amino acids withreactive side-chains such as Lys, Cys and Tyr resulted in the conversionof lysine to N²-acetyl-lysine. Chemical synthesis also provides astraightforward method to incorporate unnatural amino acids. With therecent development of enzymatic ligation and native chemical ligation ofpeptide fragments, it is possible to make larger proteins. See, e.g., P.E. Dawson and S. B. H. Kent, Annu. Rev. Biochem, 69:923 (2000). Ageneral in vitro biosynthetic method in which a suppressor tRNAchemically acylated with the desired unnatural amino acid is added to anin vitro extract capable of supporting protein biosynthesis, has beenused to site-specifically incorporate over 100 unnatural amino acidsinto a variety of proteins of virtually any size. See, e.g., V. W.Cornish, D. Mendel and P. G. Schultz, Angew. Chem. Int. Ed. Engl., 1995,34:621 (1995); C. J. Noren, S. J. Anthony-Cahill, M. C. Griffith, P. G.Schultz, A general method for site-specific incorporation of unnaturalamino acids into proteins, Science 244:182-188 (1989); and, J. D. Bain,C. G. Glabe, T. A. Dix, A. R. Chamberlin, E. S. Diala, Biosyntheticsite-specific incorporation of a non-natural amino acid into apolypeptide, J. Am. Chem. Soc. 111:8013-8014 (1989). A broad range offunctional groups has been introduced into proteins for studies ofprotein stability, protein folding, enzyme mechanism, and signaltransduction. An in vivo method, termed selective pressureincorporation, was developed to exploit the promiscuity of wild-typesynthetases. See, e.g., N. Budisa, C. Minks, S. Alefelder, W. Wenger, F.M. Dong, L. Moroder and R. Huber, FASEB J., 13:41 (1999). An auxotrophicstrain, in which the relevant metabolic pathway supplying the cell witha particular natural amino acid is switched off, is grown in minimalmedia containing limited concentrations of the natural amino acid, whiletranscription of the target gene is repressed. At the onset of astationary growth phase, the natural amino acid is depleted and replacedwith the unnatural amino acid analog. Induction of expression of therecombinant protein results in the accumulation of a protein containingthe unnatural analog. For example, using this strategy, o, m andp-fluorophenylalanines have been incorporated into proteins, and exhibittwo characteristic shoulders in the UV spectrum which can be easilyidentified, see, e.g., C. Minks, R. Huber, L. Moroder and N. Budisa,Anal. Biochem., 284:29 (2000); trifluoromethionine has been used toreplace methionine in bacteriophage T4 lysozyme to study its interactionwith chitooligosaccharide ligands by ¹⁹F NMR, see, e.g., H. Duewel, E.Daub, V. Robinson and J. F. Honek, Biochemistry, 36:3404 (1997); andtrifluoroleucine has been incorporated in place of leucine, resulting inincreased thermal and chemical stability of a leucine-zipper protein.See, e.g., Y. Tang, G. Ghirlanda, W. A. Petka, T. Nakajima, W. F.DeGrado and D. A. Tirrell, Angew. Chem. Int. Ed. Engl., 40:1494 (2001).Moreover, selenomethionine and telluromethionine are incorporated intovarious recombinant proteins to facilitate the solution of phases inX-ray crystallography. See, e.g., W. A. Hendrickson, J. R. Horton and D.M. Lemaster, EMBO J., 9:1665 (1990); J. O. Boles, K. Lewinski, M.Kunkle, J. D. Odom, B. Dunlap, L. Lebioda and M. Hatada, Nat. Struct.Biol., 1:283 (1994); N. Budisa, B. Steipe, P. Demange, C. Eckerskorn, J.Kellermann and R. Huber, Eur. J. Biochem., 230:788 (1995); and, N.Budisa, W. Karnbrock, S. Steinbacher, A. Humm, L. Prade, T. Neuefeind,L. Moroder and R. Huber, J. Mol. Biol., 270:616 (1997). Methionineanalogs with alkene or alkyne functionalities have also beenincorporated efficiently, allowing for additional modification ofproteins by chemical means. See, e.g., J. C. van Hest and D. A. Tirrell,FEBS Lett., 428:68 (1998); J. C. van Hest, K. L. Kiick and D. A.Tirrell, J. Am. Chem. Soc., 122:1282 (2000); and, K. L. Kiick and D. A.Tirrell, Tetrahedron, 56:9487 (2000); U.S. Pat. No. 6,586,207; U.S.Patent Publication 2002/0042097, which are incorporated by referenceherein.

The success of this method depends on the recognition of the unnaturalamino acid analogs by aminoacyl-tRNA synthetases, which, in general,require high selectivity to insure the fidelity of protein translation.One way to expand the scope of this method is to relax the substratespecificity of aminoacyl-tRNA synthetases, which has been achieved in alimited number of cases. For example, replacement of Ala²⁹⁴ by Gly inEscherichia coli phenylalanyl-tRNA synthetase (PheRS) increases the sizeof substrate binding pocket, and results in the acylation of tRNAPhe byp-Cl-phenylalanine (p-Cl-Phe). See, M. Ibba, P. Kast and H. Hennecke,Biochemistry, 33:7107 (1994). An Escherichia coli strain harboring thismutant PheRS allows the incorporation of p-Cl-phenylalanine orp-Br-phenylalanine in place of phenylalanine. See, e.g., M. Ibba and H.Hennecke, FEBS Lett., 364:272 (1995); and, N. Sharma, R. Furter, P. Kastand D. A. Tirrell, FEBS Lett., 467:37 (2000). Similarly, a pointmutation Phe130Ser near the amino acid binding site of Escherichia colityrosyl-tRNA synthetase was shown to allow azatyrosine to beincorporated more efficiently than tyrosine. See, F. Hamano-Takaku, T.Iwama, S. Saito-Yano, K. Takaku, Y. Monden, M. Kitabatake, D. Soll andS. Nishimura, J. Biol. Chem., 275:40324 (2000).

Another strategy to incorporate unnatural amino acids into proteins invivo is to modify synthetases that have proofreading mechanisms. Thesesynthetases cannot discriminate and therefore activate amino acids thatare structurally similar to the cognate natural amino acids. This erroris corrected at a separate site, which deacylates the mischarged aminoacid from the tRNA to maintain the fidelity of protein translation. Ifthe proofreading activity of the synthetase is disabled, structuralanalogs that are misactivated may escape the editing function and beincorporated. This approach has been demonstrated recently with thevalyl-tRNA synthetase (ValRS). See, V. Doring, H. D. Mootz, L. A.Nangle, T. L. Hendrickson, V. de Crecy-Lagard, P. Schimmel and P.Marliere, Science, 292:501 (2001). ValRS can misaminoacylate tRNAValwith Cys, Thr, or aminobutyrate (Abu); these noncognate amino acids aresubsequently hydrolyzed by the editing domain. After random mutagenesisof the Escherichia coli chromosome, a mutant Escherichia coli strain wasselected that has a mutation in the editing site of ValRS. Thisedit-defective ValRS incorrectly charges tRNAVal with Cys. Because Abusterically resembles Cys (—SH group of Cys is replaced with —CH3 inAbu), the mutant ValRS also incorporates Abu into proteins when thismutant Escherichia coli strain is grown in the presence of Abu. Massspectrometric analysis shows that about 24% of valines are replaced byAbu at each valine position in the native protein.

Solid-phase synthesis and semisynthetic methods have also allowed forthe synthesis of a number of proteins containing novel amino acids. Forexample, see the following publications and references cited within,which are as follows: Crick, F. H. C., Barrett, L. Brenner, S.Watts-Tobin, R. General nature of the genetic code for proteins. Nature,192:1227-1232 (1961); Hofmann, K., Bohn, H. Studies on polypeptides.XXXVI. The effect of pyrazole-imidazole replacements on the S-proteinactivating potency of an S-peptide fragment, J. Am. Chem,88(24):5914-5919 (1966); Kaiser, E. T. Synthetic approaches tobiologically active peptides and proteins including enyzmes, Ace ChemRes, 22:47-54 (1989); Nakatsuka, T., Sasaki, T., Kaiser, E. T. Peptidesegment coupling catalyzed by the semisynthetic enzyme thiosubtilisin, JAm Chem Soc, 109:3808-3810 (1987); Schnolzer, M., Kent, S B H.Constructing proteins by dovetailing unprotected synthetic peptides:backbone-engineered HIV protease, Science, 256(5054):221-225 (1992);Chaiken, I. M. Semisynthetic peptides and proteins, CRC Crit. RevBiochem, 11(3):255-301 (1981); Offord, R. E. Protein engineering bychemical means? Protein Eng., 1(3):151-157 (1987); and, Jackson, D. Y.,Burnier, J., Quan, C., Stanley, M., Tom, J., Wells, J. A. A DesignedPeptide Ligase for Total Synthesis of Ribonuclease A with UnnaturalCatalytic Residues, Science, 266(5183):243 (1994).

Chemical modification has been used to introduce a variety of unnaturalside chains, including cofactors, spin labels and oligonucleotides intoproteins in vitro. See, e.g., Corey, D. R., Schultz, P. G. Generation ofa hybrid sequence-specific single-stranded deoxyribonuclease, Science,238(4832):1401-1403 (1987); Kaiser, E. T., Lawrence D. S., Rokita, S. E.The chemical modification of enzymatic specificity, Annu Rev Biochem,54:565-595 (1985); Kaiser, E. T., Lawrence, D. S. Chemical mutation ofenyzme active sites, Science, 226(4674):505-511 (1984); Neet, K. E.,Nanci A, Koshland, D. E. Properties of thiol-subtilisin, J. Biol. Chem.,243(24):6392-6401 (1968); Polgar, L. et M. L. Bender. A new enzymecontaining a synthetically formed active site. Thiol-subtilisin. J. Am.Chem Soc, 88:3153-3154 (1966); and, Pollack, S. J., Nakayama, G.Schultz, P. G. Introduction of nucleophiles and spectroscopic probesinto antibody combining sites, Science, 242(4881): 1038-1040 (1988).

Alternatively, biosynthetic methods that employ chemically modifiedaminoacyl-tRNAs have been used to incorporate several biophysical probesinto proteins synthesized in vitro. See the following publications andreferences cited within: Brunner, J. New Photolabeling and crosslinkingmethods, Annu. Rev Biochem, 62:483-514 (1993); and, Krieg, U. C.,Walter, P., Hohnson, A. E. Photocrosslinking of the signal sequence ofnascent preprolactin of the 54-kilodalton polypeptide of the signalrecognition particle, Proc. Natl. Acad. Sci, 83(22):8604-8608 (1986).

Previously, it has been shown that unnatural amino acids can besite-specifically incorporated into proteins in vitro by the addition ofchemically aminoacylated suppressor tRNAs to protein synthesis reactionsprogrammed with a gene containing a desired amber nonsense mutation.Using these approaches, one can substitute a number of the common twentyamino acids with close structural homologues, e.g., fluorophenylalaninefor phenylalanine, using strains auxotropic for a particular amino acid.See, e.g., Noren, C. J., Anthony-Cahill, Griffith, M. C., Schultz, P. G.A general method for site-specific incorporation of unnatural aminoacids into proteins, Science, 244: 182-188 (1989); M. W. Nowak, et al.,Science 268:439-42 (1995); Bain, J. D., Glabe, C. G., Dix, T. A.,Chamberlin, A. R., Diala, E. S. Biosynthetic site-specific Incorporationof a non-natural amino acid into a polypeptide, J. Am. Chem Soc,111:8013-8014 (1989); N. Budisa et al., FASEB J. 13:41-51 (1999);Ellman, J. A., Mendel, D., Anthony-Cahill, S., Noren, C. J., Schultz, P.G. Biosynthetic method for introducing unnatural amino acidssite-specifically into proteins, Methods in Enz., vol. 202, 301-336(1992); and, Mendel, D., Cornish, V. W. & Schultz, P. G. Site-DirectedMutagenesis with an Expanded Genetic Code, Annu Rev Biophys. BiomolStruct. 24, 435-62 (1995).

For example, a suppressor tRNA was prepared that recognized the stopcodon UAG and was chemically aminoacylated with an unnatural amino acid.Conventional site-directed mutagenesis was used to introduce the stopcodon TAG, at the site of interest in the protein gene. See, e.g.,Sayers, J. R., Schmidt, W. Eckstein, F. 5′-3′ Exonucleases inphosphorothioate-based olignoucleotide-directed mutagensis, NucleicAcids Res, 16(3):791-802 (1988). When the acylated suppressor tRNA andthe mutant gene were combined in an in vitro transcription/translationsystem, the unnatural amino acid was incorporated in response to the UAGcodon which gave a protein containing that amino acid at the specifiedposition. Experiments using [³H]-Phe and experiments with α-hydroxyacids demonstrated that only the desired amino acid is incorporated atthe position specified by the UAG codon and that this amino acid is notincorporated at any other site in the protein. See, e.g., Noren, et al,supra; Kobayashi et al., (2003) Nature Structural Biology 10(6):425-432;and, Ellman, J. A., Mendel, D., Schultz, P. G. Site-specificincorporation of novel backbone structures into proteins, Science,255(5041):197-200 (1992).

Microinjection techniques have also been use incorporate unnatural aminoacids into proteins. See, e.g., M. W. Nowak, P. C. Kearney, J. R.Sampson, M. E. Saks, C. G. Labarca, S. K. Silverman, W. G. Zhong, J.Thorson, J. N. Abelson, N. Davidson, P. G. Schultz, D. A. Dougherty andH. A. Lester, Science, 268:439 (1995); and, D. A. Dougherty, Curr. Opin.Chem. Biol., 4:645 (2000). A Xenopus oocyte was coinjected with two RNAspecies made in vitro: an mRNA encoding the target protein with a UAGstop codon at the amino acid position of interest and an ambersuppressor tRNA aminoacylated with the desired unnatural amino acid. Thetranslational machinery of the oocyte then inserts the unnatural aminoacid at the position specified by UAG. This method has allowed in vivostructure-function studies of integral membrane proteins, which aregenerally not amenable to in vitro expression systems. Examples includethe incorporation of a fluorescent amino acid into tachykininneurokinin-2 receptor to measure distances by fluorescence resonanceenergy transfer, see, e.g., G. Turcatti, K. Nemeth, M. D. Edgerton, U.Meseth, F. Talabot, M. Peitsch, J. Knowles, H. Vogel and A. Chollet, J.Biol. Chem., 271:19991 (1996); the incorporation of biotinylated aminoacids to identify surface-exposed residues in ion channels, see, e.g.,J. P. Gallivan, H. A. Lester and D. A. Dougherty, Chem. Biol., 4:739(1997); the use of caged tyrosine analogs to monitor conformationalchanges in an ion channel in real time, see, e.g., J. C. Miller, S. K.Silverman, P. M. England, D. A. Dougherty and H. A. Lester, Neuron,20:619 (1998); and, the use of alpha hydroxy amino acids to change ionchannel backbones for probing their gating mechanisms. See, e.g., P. M.England, Y. Zhang, D. A. Dougherty and H. A. Lester, Cell, 96:89 (1999);and, T. Lu, A. Y. Ting, J. Mainland, L. Y. Jan, P. G. Schultz and J.Yang, Nat. Neurosci., 4:239 (2001).

The ability to incorporate unnatural amino acids directly into proteinsin vivo offers the advantages of high yields of mutant proteins,technical ease, the potential to study the mutant proteins in cells orpossibly in living organisms and the use of these mutant proteins intherapeutic treatments. The ability to include unnatural amino acidswith various sizes, acidities, nucleophilicities, hydrophobicities, andother properties into proteins can greatly expand our ability torationally and systematically manipulate the structures of proteins,both to probe protein function and create new proteins or organisms withnovel properties. However, the process is difficult, because the complexnature of tRNA-synthetase interactions that are required to achieve ahigh degree of fidelity in protein translation.

In one attempt to site-specifically incorporate para-F-Phe, a yeastamber suppressor tRNAPheCUA/phenylalanyl-tRNA synthetase pair was usedin a p-F-Phe resistant, Phe auxotrophic Escherichia coli strain. See,e.g., R. Furter, Protein Sci., 7:419 (1998).

It may also be possible to obtain expression of BPFI of the presentinvention using a cell-free (in-vitro) translational system. In thesesystems, which can include either mRNA as a template (in-vitrotranslation) or DNA as a template (combined in-vitro transcription andtranslation), the in vitro synthesis is directed by the ribosomes.Considerable effort has been applied to the development of cell-freeprotein expression systems. See, e.g., Kim, D. M. and J. R. Swartz,Biotechnology and Bioengineering, 74 :309-316 (2001); Kim, D. M. and J.R. Swartz, Biotechnology Letters, 22, 1537-1542, (2000); Kim, D. M., andJ. R. Swartz, Biotechnology Progress, 16, 385-390, (2000); Kim, D. M.,and J. R. Swartz, Biotechnology and Bioengineering, 66, 180-188, (1999);and Patnaik, R. and J. R. Swartz, Biotechniques 24, 862-868, (1998);U.S. Pat. No. 6,337,191; U.S. Patent Publication No. 2002/0081660; WO00/55353; WO 90/05785, which are incorporated by reference herein.Another approach that may be applied to the expression of BPFIscomprising a non-naturally encoded amino acid includes the mRNA-peptidefusion technique. See, e.g., R. Roberts and J. Szostak, Proc. Natl.Acad. Sci. (USA) 94:12297-12302 (1997); A. Frankel, et al., Chemistry &Biology 10:1043-1050 (2003). In this approach, an mRNA template linkedto puromycin is translated into peptide on the ribosome. If one or moretRNA molecules has been modified, non-natural amino acids can beincorporated into the peptide as well. After the last mRNA codon hasbeen read, puromycin captures the C-terminus of the peptide. If theresulting mRNA-peptide conjugate is found to have interesting propertiesin an in vitro assay, its identity can be easily revealed from the mRNAsequence. In this way, one may screen libraries of BPFIs comprising oneor more non-naturally encoded amino acids to identify polypeptideshaving desired properties. More recently, in vitro ribosome translationswith purified components have been reported that permit the synthesis ofpeptides substituted with non-naturally encoded amino acids. See, e.g.,A. Forster et al., Proc. Natl. Acad. Sci. (USA) 100:6353 (2003).

IX. Macromolecular Polymers Coupled to BPFI

Various modifications to the non-natural amino acid polypeptidesdescribed herein can be effected using the compositions, methods,techniques and strategies described herein. These modifications includethe incorporation of further functionality onto the non-natural aminoacid component of the polypeptide, including but not limited to, alabel; a dye; a polymer; a water-soluble polymer; a derivative ofpolyethylene glycol; a photocrosslinker; a radionuclide; cytotoxiccompound; a drug; an affinity label; a photoaffinity label; a reactivecompound; a resin; a second protein or polypeptide or polypeptideanalog; an antibody or antibody fragment; a metal chelator; a cofactor;a fatty acid; a carbohydrate; a polynucleotide; a DNA; a RNA; anantisense polynucleotide; a water-soluble dendimer; a cyclodextrin; aninhibitory ribonucleic acid; a biomaterial; a nanoparticle; a spinlabel; a fluorophore, a metal-containing moiety; a radioactive moiety; anovel functional group; a group that covalently or noncovalentlyinteracts with other molecules; a photocaged moiety; a photoisomerizablemoiety; biotin; a derivative of biotin; a biotin analogue; a moietyincorporating a heavy atom; a chemically cleavable group; aphotocleavable group; an elongated side chain; a carbon-linked sugar; aredox-active agent; an amino thioacid; a toxic moiety; an isotopicallylabeled moiety; a biophysical probe; a phosphorescent group; achemiluminescent group; an electron dense group; a magnetic group; anintercalating group; a chromophore; an energy transfer agent; abiologically active agent; a detectable label; a small molecule; or anycombination of the above, or any other desirable compound or substance.As an illustrative, non-limiting example of the compositions, methods,techniques and strategies described herein, the following descriptionwill focus on adding macromolecular polymers to the non-natural aminoacid polypeptide with the understanding that the compositions, methods,techniques and strategies described thereto are also applicable (withappropriate modifications, if necessary and for which one of skill inthe art could make with the disclosures herein) to adding otherfunctionalities, including but not limited to those listed above.

A wide variety of macromolecular polymers and other molecules can belinked to BPFIs of the present invention to modulate biologicalproperties of the BPFI, and/or provide new biological properties to theBPFI molecule. These macromolecular polymers can be linked to BPFI via anaturally encoded amino acid, via a non-naturally encoded amino acid, orany functional substituent of a natural or non-natural amino acid, orany substituent or functional group added to a natural or non-naturalamino acid. The molecular weight of the polymer may be of a wide range,including but not limited to, between about 100 Da and about 100,000 Daor more. The molecular weight of the polymer may be of a wide range,including but not limited to, between about 100 Da and about 100,000 Daor more. The molecular weight of the polymer may be between about 100 Daand about 100,000 Da, including but not limited to, 100,000 Da, 95,000Da, 90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da,60,000 Da, 55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000 Da, 30,000Da, 25,000 Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da, 8,000 Da,7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, 1,000 Da,900 Da, 800 Da, 700 Da, 600 Da, 500 Da, 400 Da, 300 Da, 200 Da, and 100Da. In some embodiments, the molecular weight of the polymer is betweenabout 100 Da and 50,000 Da. In some embodiments, the molecular weight ofthe polymer is between about 100 Da and 40,000 Da. In some embodiments,the molecular weight of the polymer is between about 1,000 Da and 40,000Da. In some embodiments, the molecular weight of the polymer is betweenabout 5,000 Da and 40,000 Da. In some embodiments, the molecular weightof the polymer is between about 10,000 Da and 40,000 Da.

The present invention provides substantially homogenous preparations ofpolymer:protein conjugates. “Substantially homogenous” as used hereinmeans that polymer:protein conjugate molecules are observed to begreater than half of the total protein. The polymer:protein conjugatehas biological activity and the present “substantially homogenous”PEGylated BPFI preparations provided herein are those which arehomogenous enough to display the advantages of a homogenous preparation,e.g., ease in clinical application in predictability of lot to lotpharmacokinetics.

One may also choose to prepare a mixture of polymer:protein conjugatemolecules, and the advantage provided herein is that one may select theproportion of mono-polymer:protein conjugate to include in the mixture.Thus, if desired, one may prepare a mixture of various proteins withvarious numbers of polymer moieties attached (i.e., di-, tri-, tetra-,etc.) and combine said conjugates with the mono-polymer:proteinconjugate prepared using the methods of the present invention, and havea mixture with a predetermined proportion of mono-polymer:proteinconjugates.

The polymer selected may be water soluble so that the protein to whichit is attached does not precipitate in an aqueous environment, such as aphysiological environment. The polymer may be branched or unbranched.Preferably, for therapeutic use of the end-product preparation, thepolymer will be pharmaceutically acceptable.

The proportion of polyethylene glycol molecules to protein moleculeswill vary, as will their concentrations in the reaction mixture. Ingeneral, the optimum ratio (in terms of efficiency of reaction in thatthere is minimal excess unreacted protein or polymer) may be determinedby the molecular weight of the polyethylene glycol selected and on thenumber of available reactive groups available. As relates to molecularweight, typically the higher the molecular weight of the polymer, thefewer number of polymer molecules which may be attached to the protein.Similarly, branching of the polymer should be taken into account whenoptimizing these parameters. Generally, the higher the molecular weight(or the more branches) the higher the polymer:protein ratio.

As used herein, and when contemplating PEG:BPFI conjugates, the term“therapeutically effective amount” refers to an amount which gives thedesired benefit to a patient. For example, the term “therapeuticallyeffective amount” refers to an amount which modulates viral level thatprovides benefit to a patient. The amount will vary from one individualto another and will depend upon a number of factors, including theoverall physical condition of the patient and the underlying cause ofthe condition to be treated. The amount of BPFI used for therapy givesan acceptable rate of change and maintains desired response at abeneficial level. A therapeutically effective amount of the presentcompositions may be readily ascertained by one skilled in the art usingpublicly available materials and procedures.

The water soluble polymer may be any structural form including but notlimited to linear, forked or branched. Typically, the water solublepolymer is a poly(alkylene glycol), such as poly(ethylene glycol) (PEG),but other water soluble polymers can also be employed. By way ofexample, PEG is used to describe certain embodiments of this invention.

PEG is a well-known, water soluble polymer that is commerciallyavailable or can be prepared by ring-opening polymerization of ethyleneglycol according to methods well known in the art (Sandler and Karo,Polymer Synthesis, Academic Press, New York, Vol. 3, pages 138-161). Theterm “PEG” is used broadly to encompass any polyethylene glycolmolecule, without regard to size or to modification at an end of thePEG, and can be represented as linked to the BPFI by the formula:

XO—(CH₂CH₂O)_(n)—CH₂CH₂—Y

where n is 2 to 10,000 and X is H or a terminal modification, includingbut not limited to, a C₁₋₄ alkyl.

In some cases, a PEG used in the invention terminates on one end withhydroxy or methoxy, i.e., X is H or CH₃ (“methoxy PEG”). Alternatively,the PEG can terminate with a reactive group, thereby forming abifunctional polymer. Typical reactive groups can include those reactivegroups that are commonly used to react with the functional groups foundin the 20 common amino acids (including but not limited to, maleimidegroups, activated carbonates (including but not limited to,p-nitrophenyl ester), activated esters (including but not limited to,N-hydroxysuccinimide, p-nitrophenyl ester) and aldehydes) as well asfunctional groups that are inert to the 20 common amino acids but thatreact specifically with complementary functional groups present innon-naturally encoded amino acids (including but not limited to, azidegroups, alkyne groups). It is noted that the other end of the PEG, whichis shown in the above formula by Y, will attach either directly orindirectly to a BPFI via a naturally-occurring or non-naturally encodedamino acid. For instance, Y may be an amide, carbamate or urea linkageto an amine group (including but not limited to, the epsilon amine oflysine or the N-terminus) of the polypeptide. Alternatively, Y may be amaleimide linkage to a thiol group (including but not limited to, thethiol group of cysteine). Alternatively, Y may be a linkage to a residuenot commonly accessible via the 20 common amino acids. For example, anazide group on the PEG can be reacted with an alkyne group on the BPFIto form a Huisgen [3+2] cycloaddition product. Alternatively, an alkynegroup on the PEG can be reacted with an azide group present in anon-naturally encoded amino acid to form a similar product. In someembodiments, a strong nucleophile (including but not limited to,hydrazine, hydrazide, hydroxylamine, semicarbazide) can be reacted withan aldehyde or ketone group present in a non-naturally encoded aminoacid to form a hydrazone, oxime or semicarbazone, as applicable, whichin some cases can be further reduced by treatment with an appropriatereducing agent. Alternatively, the strong nucleophile can beincorporated into the BPFI via a non-naturally encoded amino acid andused to react preferentially with a ketone or aldehyde group present inthe water soluble polymer.

Any molecular mass for a PEG can be used as practically desired,including but not limited to, from about 100 Daltons (Da) to 100,000 Daor more as desired (including but not limited to, sometimes 0.1-50 kDaor 10-40 kDa). Branched chain PEGs, including but not limited to, PEGmolecules with each chain having a MW ranging from 1-100 kDa (includingbut not limited to, 1-50 kDa or 5-20 kDa) can also be used. A wide rangeof PEG molecules are described in, including but not limited to, theShearwater Polymers, Inc. catalog, Nektar Therapeutics catalog,incorporated herein by reference.

Generally, at least one terminus of the PEG molecule is available forreaction with the non-naturally-encoded amino acid. For example, PEGderivatives bearing alkyne and azide moieties for reaction with aminoacid side chains can be used to attach PEG to non-naturally encodedamino acids as described herein. If the non-naturally encoded amino acidcomprises an azide, then the PEG will typically contain either an alkynemoiety to effect formation of the [3+2] cycloaddition product or anactivated PEG species (i.e., ester, carbonate) containing a phosphinegroup to effect formation of the amide linkage. Alternatively, if thenon-naturally encoded amino acid comprises an alkyne, then the PEG willtypically contain an azide moiety to effect formation of the [3+2]Huisgen cycloaddition product. If the non-naturally encoded amino acidcomprises a carbonyl group, the PEG will typically comprise a potentnucleophile (including but not limited to, a hydrazide, hydrazine,hydroxylamine, or semicarbazide functionality) in order to effectformation of corresponding hydrazone, oxime, and semicarbazone linkages,respectively. In other alternatives, a reverse of the orientation of thereactive groups described above can be used, i.e., an azide moiety inthe non-naturally encoded amino acid can be reacted with a PEGderivative containing an alkyne.

In some embodiments, the BPFI variant with a PEG derivative contains achemical functionality that is reactive with the chemical functionalitypresent on the side chain of the non-naturally encoded amino acid.

The invention provides in some embodiments azide- andacetylene-containing polymer derivatives comprising a water solublepolymer backbone having an average molecular weight from about 800 Da toabout 100,000 Da. The polymer backbone of the water-soluble polymer canbe poly(ethylene glycol). However, it should be understood that a widevariety of water soluble polymers including but not limited topoly(ethylene)glycol and other related polymers, including poly(dextran)and poly(propylene glycol), are also suitable for use in the practice ofthis invention and that the use of the term PEG or poly(ethylene glycol)is intended to encompass and include all such molecules. The term PEGincludes, but is not limited to, poly(ethylene glycol) in any of itsforms, including bifunctional PEG, multiarmed PEG, derivatized PEG,forked PEG, branched PEG, pendent PEG (i.e. PEG or related polymershaving one or more functional groups pendent to the polymer backbone),or PEG with degradable linkages therein.

PEG is typically clear, colorless, odorless, soluble in water, stable toheat, inert to many chemical agents, does not hydrolyze or deteriorate,and is generally non-toxic. Poly(ethylene glycol) is considered to bebiocompatible, which is to say that PEG is capable of coexistence withliving tissues or organisms without causing harm. More specifically, PEGis substantially non-immunogenic, which is to say that PEG does not tendto produce an immune response in the body. When attached to a moleculehaving some desirable function in the body, such as a biologicallyactive agent, the PEG tends to mask the agent and can reduce oreliminate any immune response so that an organism can tolerate thepresence of the agent. PEG conjugates tend not to produce a substantialimmune response or cause clotting or other undesirable effects. PEGhaving the formula —CH₂CH₂O—(CH₂CH₂O)_(n)CH₂CH₂—, where n is from about3 to about 4000, typically from about 20 to about 2000, is suitable foruse in the present invention. PEG having a molecular weight of fromabout 800 Da to about 100,000 Da are in some embodiments of the presentinvention particularly useful as the polymer backbone.

The polymer backbone can be linear or branched. Branched polymerbackbones are generally known in the art. Typically, a branched polymerhas a central branch core moiety and a plurality of linear polymerchains linked to the central branch core. PEG is commonly used inbranched forms that can be prepared by addition of ethylene oxide tovarious polyols, such as glycerol, glycerol oligomers, pentaerythritoland sorbitol. The central branch moiety can also be derived from severalamino acids, such as lysine. The branched poly(ethylene glycol) can berepresented in general form as R(—PEG-OH)_(m) in which R is derived froma core moiety, such as glycerol, glycerol oligomers, or pentaerythritol,and m represents the number of arms. Multi-armed PEG molecules, such asthose described in U.S. Pat. Nos. 5,932,462 5,643,575; 5,229,490;4,289,872; U.S. Pat. Appl. 2003/0143596; WO 96/21469; and WO 93/21259,each of which is incorporated by reference herein in its entirety, canalso be used as the polymer backbone.

Branched PEG can also be in the form of a forked PEG represented byPEG(—YCHZ₂)_(n), where Y is a linking group and Z is an activatedterminal group linked to CH by a chain of atoms of defined length.

Yet another branched form, the pendant PEG, has reactive groups, such ascarboxyl, along the PEG backbone rather than at the end of PEG chains.

In addition to these forms of PEG, the polymer can also be prepared withweak or degradable linkages in the backbone. For example, PEG can beprepared with ester linkages in the polymer backbone that are subject tohydrolysis. As shown below, this hydrolysis results in cleavage of thepolymer into fragments of lower molecular weight:

-PEG-CO₂-PEG-+H₂O→PEG-CO₂H+HO-PEG-

It is understood by those skilled in the art that the term poly(ethyleneglycol) or PEG represents or includes all the forms known in the artincluding but not limited to those disclosed herein.

Many other polymers are also suitable for use in the present invention.In some embodiments, polymer backbones that are water-soluble, with from2 to about 300 termini, are particularly useful in the invention.Examples of suitable polymers include, but are not limited to, otherpoly(alkylene glycols), such as poly(propylene glycol) (“PPG”),copolymers thereof (including but not limited to copolymers of ethyleneglycol and propylene glycol), terpolymers thereof, mixtures thereof, andthe like. Although the molecular weight of each chain of the polymerbackbone can vary, it is typically in the range of from about 800 Da toabout 100,000 Da, often from about 6,000 Da to about 80,000 Da.

Those of ordinary skill in the art will recognize that the foregoinglist for substantially water soluble backbones is by no means exhaustiveand is merely illustrative, and that all polymeric materials having thequalities described above are contemplated as being suitable for use inthe present invention.

In some embodiments of the present invention the polymer derivatives are“multi-functional”, meaning that the polymer backbone has at least twotermini, and possibly as many as about 300 termini, functionalized oractivated with a functional group. Multifunctional polymer derivativesinclude, but are not limited to, linear polymers having two termini,each terminus being bonded to a functional group which may be the sameor different.

In one embodiment, the polymer derivative has the structure:

X-A-POLY-B—N═N═N

wherein:N═N═N is an azide moiety;B is a linking moiety, which may be present or absent;POLY is a water-soluble non-antigenic polymer;A is a linking moiety, which may be present or absent and which may bethe same as B or different; andX is a second functional group.Examples of a linking moiety for A and B include, but are not limitedto, a multiply-functionalized alkyl group containing up to 18, and morepreferably between 1-10 carbon atoms. A heteroatom such as nitrogen,oxygen or sulfur may be included with the alkyl chain. The alkyl chainmay also be branched at a heteroatom. Other examples of a linking moietyfor A and B include, but are not limited to, a multiply functionalizedaryl group, containing up to 10 and more preferably 5-6 carbon atoms.The aryl group may be substituted with one more carbon atoms, nitrogen,oxygen or sulfur atoms. Other examples of suitable linking groupsinclude those linking groups described in U.S. Pat. Nos. 5,932,462;5,643,575; and U.S. Pat. Appl. Publication 2003/0143596, each of whichis incorporated by reference herein. Those of ordinary skill in the artwill recognize that the foregoing list for linking moieties is by nomeans exhaustive and is merely illustrative, and that all linkingmoieties having the qualities described above are contemplated to besuitable for use in the present invention.

Examples of suitable functional groups for use as X include, but are notlimited to, hydroxyl, protected hydroxyl, alkoxyl, active ester, such asN-hydroxysuccinimidyl esters and 1-benzotriazolyl esters, activecarbonate, such as N-hydroxysuccinimidyl carbonates and 1-benzotriazolylcarbonates, acetal, aldehyde, aldehyde hydrates, alkenyl, acrylate,methacrylate, acrylamide, active sulfone, amine, aminooxy, protectedamine, hydrazide, protected hydrazide, protected thiol, carboxylic acid,protected carboxylic acid, isocyanate, isothiocyanate, maleimide,vinylsulfone, dithiopyridine, vinylpyridine, iodoacetamide, epoxide,glyoxals, diones, mesylates, tosylates, tresylate, alkene, ketone, andazide. As is understood by those skilled in the art, the selected Xmoiety should be compatible with the azide group so that reaction withthe azide group does not occur. The azide-containing polymer derivativesmay be homobifunctional, meaning that the second functional group (i.e.,X) is also an azide moiety, or heterobifunctional, meaning that thesecond functional group is a different functional group.

The term “protected” refers to the presence of a protecting group ormoiety that prevents reaction of the chemically reactive functionalgroup under certain reaction conditions. The protecting group will varydepending on the type of chemically reactive group being protected. Forexample, if the chemically reactive group is an amine or a hydrazide,the protecting group can be selected from the group oftert-butyloxycarbonyl (t-Boc) and 9-fluorenylmethoxycarbonyl (Fmoc). Ifthe chemically reactive group is a thiol, the protecting group can beorthopyridyldisulfide. If the chemically reactive group is a carboxylicacid, such as butanoic or propionic acid, or a hydroxyl group, theprotecting group can be benzyl or an alkyl group such as methyl, ethyl,or tert-butyl. Other protecting groups known in the art may also be usedin the present invention.

Specific examples of terminal functional groups in the literatureinclude, but are not limited to, N-succinimidyl carbonate (see e.g.,U.S. Pat. Nos. 5,281,698, 5,468,478), amine (see, e.g., Buckmann et al.Makromol. Chem. 182:1379 (1981), Zalipsky et al. Eur. Polym. J. 19:1177(1983)), hydrazide (See, e.g., Andresz et al. Makromol. Chem. 179:301(1978)), succinimidyl propionate and succinimidyl butanoate (see, e.g.,Olson et al. in Poly(ethylene glycol) Chemistry & BiologicalApplications, pp 170-181, Harris & Zalipsky Eds., ACS, Washington, D.C.,1997; see also U.S. Pat. No. 5,672,662), succinimidyl succinate (See,e.g., Abuchowski et al. Cancer Biochem. Biophys. 7:175 (1984) andJoppich et al. Makrolol. Chem. 180:1381 (1979), succinimidyl ester (see,e.g., U.S. Pat. No. 4,670,417), benzotriazole carbonate (see, e.g., U.S.Pat. No. 5,650,234), glycidyl ether (see, e.g., Pitha et al. Eur. J.Biochem. 94:11 (1979), Elling et al., Biotech. Appl. Biochem. 13:354(1991), oxycarbonylimidazole (see, e.g., Beauchamp, et al., Anal.Biochem. 131:25 (1983), Tondelli et al. J. Controlled Release 1:251(1985)), p-nitrophenyl carbonate (see, e.g., Veronese, et al., Appl.Biochem. Biotech., 11: 141 (1985); and Sartore et al., Appl. Biochem.Biotech., 27:45 (1991)), aldehyde (see, e.g., Harris et al. J. Polym.Sci. Chem. Ed. 22:341 (1984), U.S. Pat. No. 5,824,784, U.S. Pat. No.5,252,714), maleimide (see, e.g., Goodson et al. Biotechnology (NY)8:343 (1990), Romani et al. in Chemistry of Peptides and Proteins 2:29(1984)), and Kogan, Synthetic Comm. 22:2417 (1992)),orthopyridyl-disulfide (see, e.g., Woghiren, et al. Bioconj. Chem. 4:314(1993)), acrylol (see, e.g., Sawhney et al., Macromolecules, 26:581(1993)), vinylsulfone (see, e.g., U.S. Pat. No. 5,900,461). All of theabove references and patents are incorporated herein by reference.

In certain embodiments of the present invention, the polymer derivativesof the invention comprise a polymer backbone having the structure:

X—CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—N═N═N

wherein:X is a functional group as described above; andn is about 20 to about 4000.In another embodiment, the polymer derivatives of the invention comprisea polymer backbone having the structure:

X—CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—O—(CH₂)_(m)—W—N═N═N

wherein:W is an aliphatic or aromatic linker moiety comprising between 1-10carbon atoms;n is about 20 to about 4000; andX is a functional group as described above. m is between 1 and 10.

The azide-containing PEG derivatives of the invention can be prepared bya variety of methods known in the art and/or disclosed herein. In onemethod, shown below, a water soluble polymer backbone having an averagemolecular weight from about 800 Da to about 100,000 Da, the polymerbackbone having a first terminus bonded to a first functional group anda second terminus bonded to a suitable leaving group, is reacted with anazide anion (which may be paired with any of a number of suitablecounter-ions, including sodium, potassium, tert-butylammonium and soforth). The leaving group undergoes a nucleophilic displacement and isreplaced by the azide moiety, affording the desired azide-containing PEGpolymer.

X-PEG-L+N₃ ⁻→X-PEG-N₃

As shown, a suitable polymer backbone for use in the present inventionhas the formula X-PEG-L, wherein PEG is poly(ethylene glycol) and X is afunctional group which does not react with azide groups and L is asuitable leaving group. Examples of suitable functional groups include,but are not limited to, hydroxyl, protected hydroxyl, acetal, alkenyl,amine, aminooxy, protected amine, protected hydrazide, protected thiol,carboxylic acid, protected carboxylic acid, maleimide, dithiopyridine,and vinylpyridine, and ketone. Examples of suitable leaving groupsinclude, but are not limited to, chloride, bromide, iodide, mesylate,tresylate, and tosylate.

In another method for preparation of the azide-containing polymerderivatives of the present invention, a linking agent bearing an azidefunctionality is contacted with a water soluble polymer backbone havingan average molecular weight from about 800 Da to about 100,000 Da,wherein the linking agent bears a chemical functionality that will reactselectively with a chemical functionality on the PEG polymer, to form anazide-containing polymer derivative product wherein the azide isseparated from the polymer backbone by a linking group.

An exemplary reaction scheme is shown below:

X-PEG-M+N-linker-N═N═N PG-X-PEG-linker-N═N═N

wherein:PEG is poly(ethylene glycol) and X is a capping group such as alkoxy ora functional group as described above; andM is a functional group that is not reactive with the azidefunctionality but that will react efficiently and selectively with the Nfunctional group.

Examples of suitable functional groups include, but are not limited to,M being a carboxylic acid, carbonate or active ester if N is an amine; Mbeing a ketone if N is a hydrazide or aminooxy moiety; M being a leavinggroup if N is a nucleophile.

Purification of the crude product may be accomplished by known methodsincluding, but are not limited to, precipitation of the product followedby chromatography, if necessary.

A more specific example is shown below in the case of PEG diamine, inwhich one of the amines is protected by a protecting group moiety suchas tert-butyl-Boc and the resulting mono-protected PEG diamine isreacted with a linking moiety that bears the azide functionality:

BocHN-PEG-NH₂+HO₂C—(CH₂)₃—N═N═N

In this instance, the amine group can be coupled to the carboxylic acidgroup using a variety of activating agents such as thionyl chloride orcarbodiimide reagents and N-hydroxysuccinimide or N-hydroxybenzotriazoleto create an amide bond between the monoamine PEG derivative and theazide-bearing linker moiety. After successful formation of the amidebond, the resulting N-tert-butyl-Boc-protected azide-containingderivative can be used directly to modify bioactive molecules or it canbe further elaborated to install other useful functional groups. Forinstance, the N-t-Boc group can be hydrolyzed by treatment with strongacid to generate an omega-amino-PEG-azide. The resulting amine can beused as a synthetic handle to install other useful functionality such asmaleimide groups, activated disulfides, activated esters and so forthfor the creation of valuable heterobifunctional reagents.

Heterobifunctional derivatives are particularly useful when it isdesired to attach different molecules to each terminus of the polymer.For example, the omega-N-amino-N-azido PEG would allow the attachment ofa molecule having an activated electrophilic group, such as an aldehyde,ketone, activated ester, activated carbonate and so forth, to oneterminus of the PEG and a molecule having an acetylene group to theother terminus of the PEG.

In another embodiment of the invention, the polymer derivative has thestructure:

X-A-POLY-B—C≡C—R

wherein:

R can be either H or an alkyl, alkene, alkyoxy, or aryl or substitutedaryl group;B is a linking moiety, which may be present or absent;POLY is a water-soluble non-antigenic polymer;A is a linking moiety, which may be present or absent and which may bethe same as B or different; andX is a second functional group.

Examples of a linking moiety for A and B include, but are not limitedto, a multiply-functionalized alkyl group containing up to 18, and morepreferably between 1-10 carbon atoms. A heteroatom such as nitrogen,oxygen or sulfur may be included with the alkyl chain. The alkyl chainmay also be branched at a heteroatom. Other examples of a linking moietyfor A and B include, but are not limited to, a multiply functionalizedaryl group, containing up to 10 and more preferably 5-6 carbon atoms.The aryl group may be substituted with one more carbon atoms, nitrogen,oxygen, or sulfur atoms. Other examples of suitable linking groupsinclude those linking groups described in U.S. Pat. Nos. 5,932,462 and5,643,575 and U.S. Pat. Appl. Publication 2003/0143596, each of which isincorporated by reference herein. Those of ordinary skill in the artwill recognize that the foregoing list for linking moieties is by nomeans exhaustive and is intended to be merely illustrative, and that awide variety of linking moieties having the qualities described aboveare contemplated to be useful in the present invention.

Examples of suitable functional groups for use as X include hydroxyl,protected hydroxyl, alkoxyl, active ester, such as N-hydroxysuccinimidylesters and 1-benzotriazolyl esters, active carbonate, such asN-hydroxysuccinimidyl carbonates and 1-benzotriazolyl carbonates,acetal, aldehyde, aldehyde hydrates, alkenyl, acrylate, methacrylate,acrylamide, active sulfone, amine, aminooxy, protected amine, hydrazide,protected hydrazide, protected thiol, carboxylic acid, protectedcarboxylic acid, isocyanate, isothiocyanate, maleimide, vinylsulfone,dithiopyridine, vinylpyridine, iodoacetamide, epoxide, glyoxals, diones,mesylates, tosylates, and tresylate, alkene, ketone, and acetylene. Aswould be understood, the selected X moiety should be compatible with theacetylene group so that reaction with the acetylene group does notoccur. The acetylene-containing polymer derivatives may behomobifunctional, meaning that the second functional group (i.e., X) isalso an acetylene moiety, or heterobifunctional, meaning that the secondfunctional group is a different functional group.

In another embodiment of the present invention, the polymer derivativescomprise a polymer backbone having the structure:

X—CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—O—(CH₂)_(m)—C≡CH

wherein:X is a functional group as described above;n is about 20 to about 4000; andm is between 1 and 10.Specific examples of each of the heterobifunctional PEG polymers areshown below.

The acetylene-containing PEG derivatives of the invention can beprepared using methods known to those skilled in the art and/ordisclosed herein. In one method, a water soluble polymer backbone havingan average molecular weight from about 800 Da to about 100,000 Da, thepolymer backbone having a first terminus bonded to a first functionalgroup and a second terminus bonded to a suitable nucleophilic group, isreacted with a compound that bears both an acetylene functionality and aleaving group that is suitable for reaction with the nucleophilic groupon the PEG. When the PEG polymer bearing the nucleophilic moiety and themolecule bearing the leaving group are combined, the leaving groupundergoes a nucleophilic displacement and is replaced by thenucleophilic moiety, affording the desired acetylene-containing polymer.

X-PEG-Nu+L-A-C→X-PEG-Nu-A-C≡CR′

As shown, a preferred polymer backbone for use in the reaction has theformula X-PEG-Nu, wherein PEG is poly(ethylene glycol), Nu is anucleophilic moiety and X is a functional group that does not react withNu, L or the acetylene functionality.

Examples of Nu include, but are not limited to, amine, alkoxy, aryloxy,sulfhydryl, imino, carboxylate, hydrazide, aminoxy groups that wouldreact primarily via a SN2-type mechanism. Additional examples of Nugroups include those functional groups that would react primarily via annucleophilic addition reaction. Examples of L groups include chloride,bromide, iodide, mesylate, tresylate, and tosylate and other groupsexpected to undergo nucleophilic displacement as well as ketones,aldehydes, thioesters, olefins, alpha-beta unsaturated carbonyl groups,carbonates and other electrophilic groups expected to undergo additionby nucleophiles.

In another embodiment of the present invention, A is an aliphatic linkerof between 1-10 carbon atoms or a substituted aryl ring of between 6-14carbon atoms. X is a functional group which does not react with azidegroups and L is a suitable leaving group

In another method for preparation of the acetylene-containing polymerderivatives of the invention, a PEG polymer having an average molecularweight from about 800 Da to about 100,000 Da, bearing either a protectedfunctional group or a capping agent at one terminus and a suitableleaving group at the other terminus is contacted by an acetylene anion.

An exemplary reaction scheme is shown below:

X-PEG-L+—C≡CR′→X-PEG-C≡CR′

wherein:PEG is poly(ethylene glycol) and X is a capping group such as alkoxy ora functional group as described above; andR′ is either H, an alkyl, alkoxy, aryl or aryloxy group or a substitutedalkyl, alkoxyl, aryl or aryloxy group.

In the example above, the leaving group L should be sufficientlyreactive to undergo SN2-type displacement when contacted with asufficient concentration of the acetylene anion. The reaction conditionsrequired to accomplish SN2 displacement of leaving groups by acetyleneanions are well known in the art.

Purification of the crude product can usually be accomplished by methodsknown in the art including, but are not limited to, precipitation of theproduct followed by chromatography, if necessary.

Water soluble polymers can be linked to BPFIs of the invention. Thewater soluble polymers may be linked via a non-naturally encoded aminoacid incorporated in the BPFI or any functional group or substituent ofa non-naturally encoded or naturally encoded amino acid, or anyfunctional group or substituent added to a non-naturally encoded ornaturally encoded amino acid. Alternatively, the water soluble polymersare linked to a BPFI incorporating a non-naturally encoded amino acidvia a naturally-occurring amino acid (including but not limited to,cysteine, lysine or the amine group of the N-terminal residue). In somecases, the BPFIs of the invention comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10non-natural amino acids, wherein one or more non-naturally-encoded aminoacid(s) are linked to water soluble polymer(s) (including but notlimited to, PEG and/or oligosaccharides). In some cases, the BPFIs ofthe invention further comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or morenaturally-encoded amino acid(s) linked to water soluble polymers. Insome cases, the BPFIs of the invention comprise one or morenon-naturally encoded amino acid(s) linked to water soluble polymers andone or more naturally-occurring amino acids linked to water solublepolymers. In some embodiments, the water soluble polymers used in thepresent invention enhance the serum half-life of the BPFI relative tothe unconjugated form.

The number of water soluble polymers linked to a BPFI (i.e., the extentof PEGylation or glycosylation) of the present invention can be adjustedto provide an altered (including but not limited to, increased ordecreased) pharmacologic, pharmacokinetic or pharmacodynamiccharacteristic such as in vivo half-life. In some embodiments, thehalf-life of BPFI is increased at least about 10, 20, 30, 40, 50, 60,70, 80, 90 percent, 2- fold, 5-fold, 10-fold, 50-fold, or at least about100-fold over an unmodified polypeptide.

PEG Derivatives Containing a Strong Nucleophilic Group (I.E., Hydrazide,Hydrazine, Hydroxylamine or Semicarbazide)

In one embodiment of the present invention, a BPFI comprising acarbonyl-containing non-naturally encoded amino acid is modified with aPEG derivative that contains a terminal hydrazine, hydroxylamine,hydrazide or semicarbazide moiety that is linked directly to the PEGbackbone.

In some embodiments, the hydroxylamine-terminal PEG derivative will havethe structure:

RO—(CH₂CH₂O)_(n)—O—(CH₂)_(m)—O—NH₂

where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and nis 100-1,000 (i.e., average molecular weight is between 5-40 kDa).

In some embodiments, the hydrazine- or hydrazide-containing PEGderivative will have the structure:

RO—(CH₂CH₂O)_(n)—O—(CH₂)_(m)—X—NH—NH₂

where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and nis 100-1,000 and X is optionally a carbonyl group (C═O) that can bepresent or absent.

In some embodiments, the semicarbazide-containing PEG derivative willhave the structure:

RO—(CH₂CH₂O)_(n)—O—(CH₂)_(m)—NH—C(O)—NH—NH₂

where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and nis 100-1,000.

In another embodiment of the invention, a BPFI comprising acarbonyl-containing amino acid is modified with a PEG derivative thatcontains a terminal hydroxylamine, hydrazide, hydrazine, orsemicarbazide moiety that is linked to the PEG backbone by means of anamide linkage.

In some embodiments, the hydroxylamine-terminal PEG derivatives have thestructure:

RO—(CH₂CH₂O)_(n)—O—(CH₂)₂—NH—C(O)(CH₂)_(m)—O—NH₂

where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and nis 100-1,000 (i.e., average molecular weight is between 5-40 kDa).

In some embodiments, the hydrazine- or hydrazide-containing PEGderivatives have the structure:

RO—(CH₂CH₂O)_(n)—O—(CH₂)₂—NH—C(O)(CH₂)_(m)—X—NH—NH₂

where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10, n is100-1,000 and X is optionally a carbonyl group (C═O) that can be presentor absent.

In some embodiments, the semicarbazide-containing PEG derivatives havethe structure:

RO—(CH₂CH₂O)_(n)—O—(CH₂)₂—NH—C(O)(CH₂)_(m)—NH—C(O)—NH—NH₂

where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and nis 100-1,000.

In another embodiment of the invention, a BPFI comprising acarbonyl-containing amino acid is modified with a branched PEGderivative that contains a terminal hydrazine, hydroxylamine, hydrazideor semicarbazide moiety, with each chain of the branched PEG having a MWranging from 10-40 kDa and, more preferably, from 5-20 kDa.

In another embodiment of the invention, a BPFI comprising anon-naturally encoded amino acid is modified with a PEG derivativehaving a branched structure. For instance, in some embodiments, thehydrazine- or hydrazide-terminal PEG derivative will have the followingstructure:

[RO—(CH₂CH₂O)_(n)—O—(CH₂)₂—NH—C(O)]₂CH(CH₂)_(m)—X—NH—NH₂

where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and nis 100-1,000, and X is optionally a carbonyl group (C═O) that can bepresent or absent.

In some embodiments, the PEG derivatives containing a semicarbazidegroup will have the structure:

[RO—(CH₂CH₂O)_(n)—O—(CH₂)₂—C(O)—NH—CH₂—CH₂]₂CH—X—(CH₂)_(m),—NH—C(O)—NH—NH₂

where R is a simple alkyl (methyl, ethyl, propyl, etc.), X is optionallyNH, O, S, C(O) or not present, m is 2-10 and n is 100-1,000.

In some embodiments, the PEG derivatives containing a hydroxylaminegroup will have the structure:

[RO—(CH₂CH₂O)_(n)—O—(CH₂)₂—C(O)—NH—CH₂—CH₂]₂CH—X—(CH₂)_(m)—O—NH₂

where R is a simple alkyl (methyl, ethyl, propyl, etc.), X is optionallyNH, O, S, C(O) or not present, m is 2-10 and n is 100-1,000.

The degree and sites at which the water soluble polymer(s) are linked tothe BPFI can modulate the binding of the BPFI to the BPFI receptor orbinding partner. In some embodiments, the linkages are arranged suchthat the BPFI binds the BPFI receptor with a K_(d) of about 400 nM orlower, with a K_(d) of 150 nM or lower, and in some cases with a K_(d)of 100 nM or lower, as measured by an equilibrium binding assay.

Methods and chemistry for activation of polymers as well as forconjugation of peptides are described in the literature and are known inthe art. Commonly used methods for activation of polymers include, butare not limited to, activation of functional groups with cyanogenbromide, periodate, glutaraldehyde, biepoxides, epichlorohydrin,divinylsulfone, carbodiimide, sulfonyl halides, trichlorotriazine, etc.(see, R. F. Taylor, (1991), PROTEIN IMMOBILISATION. FUNDAMENTAL ANDAPPLICATIONS, Marcel Dekker, N.Y.; S. S. Wong, (1992), CHEMISTRY OFPROTEIN CONJUGATION AND CROSSLINKING, CRC Press, Boca Raton; G. T.Hermanson et al., (1993), IMMOBILIZED AFFINITY LIGAND TECHNIQUES,Academic Press, N.Y.; Dunn, R. L., et al., Eds. POLYMERIC DRUGS AND DRUGDELIVERY SYSTEMS, ACS Symposium Series Vol. 469, American ChemicalSociety, Washington, D.C. 1991).

Several reviews and monographs on the functionalization and conjugationof PEG are available. See, for example, Harris, Macromol. Chem. Phys.C25: 325-373 (1985); Scouten, Methods in Enzymology 135: 30-65 (1987);Wong et al., Enzyme Microb. Technol. 14: 866-874 (1992); Delgado et al.,Critical Reviews in Therapeutic Drug Carrier Systems 9: 249-304 (1992);Zalipsky, Bioconjugate Chem. 6: 150-165 (1995).

Methods for activation of polymers can also be found in WO 94/17039,U.S. Pat. No. 5,324,844, WO 94/18247, WO 94/04193, U.S. Pat. No.5,219,564, U.S. Pat. No. 5,122,614, WO 90/13540, U.S. Pat. No.5,281,698, and WO 93/15189, and for conjugation between activatedpolymers and enzymes including but not limited to Coagulation FactorVIII (WO 94/15625), hemoglobin (WO 94/09027), oxygen carrying molecule(U.S. Pat. No. 4,412,989), ribonuclease and superoxide dismutase(Veronese at al., App. Biochem. Biotech. 11: 141-52 (1985)). Allreferences and patents cited are incorporated by reference herein.

PEGylation (i.e., addition of any water soluble polymer) of BPFIscontaining a non-naturally encoded amino acid, such asp-azido-L-phenylalanine, is carried out by any convenient method. Forexample, BPFI is PEGylated with an alkyne-terminated mPEG derivative.Briefly, an excess of solid mPEG(5000)-O—CH₂—C≡CH is added, withstirring, to an aqueous solution of p-azido-L-Phe-containing BPFI atroom temperature. Typically, the aqueous solution is buffered with abuffer having a pK_(a) near the pH at which the reaction is to becarried out (generally about pH 4-10). Examples of suitable buffers forPEGylation at pH 7.5, for instance, include, but are not limited to,HEPES, phosphate, borate, TRIS-HCl, EPPS, and TES. The pH iscontinuously monitored and adjusted if necessary. The reaction istypically allowed to continue for between about 1-48 hours.

The reaction products are subsequently subjected to hydrophobicinteraction chromatography to separate the PEGylated BPFI variants fromfree mPEG(5000)-O—CH₂—C≡CH and any high-molecular weight complexes ofthe pegylated BPFI which may form when unblocked PEG is activated atboth ends of the molecule, thereby crosslinking BPFI variant molecules.The conditions during hydrophobic interaction chromatography are suchthat free mPEG(5000)-O—CH₂—C≡CH flows through the column, while anycrosslinked PEGylated BPFI variant complexes elute after the desiredforms, which contain one BPFI variant molecule conjugated to one or morePEG groups. Suitable conditions vary depending on the relative sizes ofthe cross-linked complexes versus the desired conjugates and are readilydetermined by those skilled in the art. The eluent containing thedesired conjugates is concentrated by ultrafiltration and desalted bydiafiltration.

If necessary, the PEGylated BPFI obtained from the hydrophobicchromatography can be purified further by one or more procedures knownto those skilled in the art including, but are not limited to, affinitychromatography; anion- or cation-exchange chromatography (using,including but not limited to, DEAE SEPHAROSE); chromatography on silica;reverse phase HPLC; gel filtration (using, including but not limited to,SEPHADEX G-75); hydrophobic interaction chromatography; size-exclusionchromatography, metal-chelate chromatography;ultrafiltration/diafiltration; ethanol precipitation; ammonium sulfateprecipitation; chromatofocusing; displacement chromatography;electrophoretic procedures (including but not limited to preparativeisoelectric focusing), differential solubility (including but notlimited to ammonium sulfate precipitation), or extraction. Apparentmolecular weight may be estimated by GPC by comparison to globularprotein standards (Preneta, Ariz. in PROTEIN PURIFICATION METHODS, APRACTICAL APPROACH (Harris & Angal, Eds.) IRL Press 1989, 293-306). Thepurity of the BPFI-PEG conjugate can be assessed by proteolyticdegradation (including but not limited to, trypsin cleavage) followed bymass spectrometry analysis. Pepinsky RB., et al., J. Pharmcol. & Exp.Ther. 297(3):1059-66 (2001).

A water soluble polymer linked to an amino acid of a BPFI of theinvention can be further derivatized or substituted without limitation.

Azide-Containing PEG Derivatives

In another embodiment of the invention, a BPFI is modified with a PEGderivative that contains an azide moiety that will react with an alkynemoiety present on the side chain of the non-naturally encoded aminoacid. In general, the PEG derivatives will have an average molecularweight ranging from 1-100 kDa and, in some embodiments, from 10-40 kDa.

In some embodiments, the azide-terminal PEG derivative will have thestructure:

RO—(CH₂CH₂O)_(n)—O—(CH₂)_(m)—N₃

where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and nis 100-1,000 (i.e., average molecular weight is between 5-40 kDa).

In another embodiment, the azide-terminal PEG derivative will have thestructure:

RO—(CH₂CH₂O)_(n)—O—(CH₂)_(m)—NH—C(O)—(CH₂)_(p)—N₃

where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10, p is2-10 and n is 100-1,000 (i.e., average molecular weight is between 5-40kDa).

In another embodiment of the invention, a BPFI comprising aalkyne-containing amino acid is modified with a branched PEG derivativethat contains a terminal azide moiety, with each chain of the branchedPEG having a MW ranging from 10-40 kDa and, more preferably, from 5-20kDa. For instance, in some embodiments, the azide-terminal PEGderivative will have the following structure:

[RO—(CH₂CH₂O)_(n)—O—(CH₂)₂—NH—C(O)]₂CH(CH₂)_(m)—X—(CH₂)_(p)N₃

where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10, p is2-10, and n is 100-1,000, and X is optionally an O, N, S or carbonylgroup (C═O), in each case that can be present or absent.

Alkyne-Containing PEG Derivatives

In another embodiment of the invention, a BPFI is modified with a PEGderivative that contains an alkyne moiety that will react with an azidemoiety present on the side chain of the non-naturally encoded aminoacid.

In some embodiments, the alkyne-terminal PEG derivative will have thefollowing structure:

RO—(CH₂CH₂O)_(n)—O—(CH₂)_(m)—C≡CH

where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and nis 100-1,000 (i.e., average molecular weight is between 5-40 kDa).

In another embodiment of the invention, a BPFI comprising analkyne-containing non-naturally encoded amino acid is modified with aPEG derivative that contains a terminal azide or terminal alkyne moietythat is linked to the PEG backbone by means of an amide linkage.

In some embodiments, the alkyne-terminal PEG derivative will have thefollowing structure:

RO—(CH₂CH₂O)_(n)—O—(CH₂)_(m)—NH—C(O)—(CH₂)_(p)—C≡CH

where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10, p is2-10 and n is 100-1,000.

In another embodiment of the invention, a BPFI comprising anazide-containing amino acid is modified with a branched PEG derivativethat contains a terminal alkyne moiety, with each chain of the branchedPEG having a MW ranging from 10-40 kDa and, more preferably, from 5-20kDa. For instance, in some embodiments, the alkyne-terminal PEGderivative will have the following structure:

[RO—(CH₂CH₂O)_(n)—O—(CH₂)₂—NH—C(O)]₂CH(CH₂)_(m)—X—(CH₂)_(p)C≡CH

where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10, p is2-10, and n is 100-1,000, and X is optionally an O, N, S or carbonylgroup (C═O), or not present.

Phosphine-Containing PEG Derivatives

In another embodiment of the invention, a BPFI is modified with a PEGderivative that contains an activated functional group (including butnot limited to, ester, carbonate) further comprising an aryl phosphinegroup that will react with an azide moiety present on the side chain ofthe non-naturally encoded amino acid. In general, the PEG derivativeswill have an average molecular weight ranging from 1-100 kDa and, insome embodiments, from 10-40 kDa.

In some embodiments, the PEG derivative will have the structure:

wherein n is 1-10; X can be O, N, S or not present, Ph is phenyl, and Wis a water soluble polymer.

In some embodiments, the PEG derivative will have the structure:

wherein X can be O, N, S or not present, Ph is phenyl, W is a watersoluble polymer and R can be H, alkyl, aryl, substituted alkyl andsubstituted aryl groups. Exemplary R groups include but are not limitedto —CH₂, —C(CH₃)₃, —OR′, —NR′R″, —SR′, -halogen, —C(O)R′, —CONR′R″,—S(O)₂R′, —S(O)₂NR′R″, —CN and —NO₂. R′, R″, R′″ and R″″ eachindependently refer to hydrogen, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, including but notlimited to, aryl substituted with 1-3 halogens, substituted orunsubstituted alkyl, alkoxy or thioalkoxy groups, or arylalkyl groups.When a compound of the invention includes more than one R group, forexample, each of the R groups is independently selected as are each R′,R″, R′″ and R″″ groups when more than one of these groups is present.When R′ and R″ are attached to the same nitrogen atom, they can becombined with the nitrogen atom to form a 5-, 6-, or 7-membered ring.For example, —NR′R″ is meant to include, but not be limited to,1-pyrrolidinyl and 4-morpholinyl. From the above discussion ofsubstituents, one of skill in the art will understand that the term“alkyl” is meant to include groups including carbon atoms bound togroups other than hydrogen groups, such as haloalkyl (including but notlimited to, —CF₃ and —CH₂CF₃) and acyl (including but not limited to,—C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and the like).

Other PEG Derivatives and General PEGylation Techniques

Other exemplary PEG molecules that may be linked to BPFIs, as well asPEGylation methods include those described in, e.g., U.S. PatentPublication No. 2004/0001838; 2002/0052009; 2003/0162949; 2004/0013637;2003/0228274; 2003/0220447; 2003/0158333; 2003/0143596; 2003/0114647;2003/0105275; 2003/0105224; 2003/0023023; 2002/0156047; 2002/0099133;2002/0086939; 2002/0082345; 2002/0072573; 2002/0052430; 2002/0040076;2002/0037949; 2002/0002250; 2001/0056171; 2001/0044526; 2001/0027217;2001/0021763; U.S. Pat. Nos. 6,646,110; 5,824,778; 5,476,653; 5,219,564;5,629,384; 5,736,625; 4,902,502; 5,281,698; 5,122,614; 5,473,034;5,516,673; 5,382,657; 6,552,167; 6,610,281; 6,515,100; 6,461,603;6,436,386; 6,214,966; 5,990,237; 5,900,461; 5,739,208; 5,672,662;5,446,090; 5,808,096; 5,612,460; 5,324,844; 5,252,714; 6,420,339;6,201,072; 6,451,346; 6,306,821; 5,559,213; 5,747,646; 5,834,594;5,849,860; 5,980,948; 6,004,573; 6,129,912; WO 97/32607, EP 229,108, EP402,378, WO 92/16555, WO 94/04193, WO 94/14758, WO 94/17039, WO94/18247, WO 94/28024, WO 95/00162, WO 95/11924, WO95/13090, WO95/33490, WO 96/00080, WO 97/18832, WO 98/41562, WO 98/48837, WO99/32134, WO 99/32139, WO 99/32140, WO 96/40791, WO 98/32466, WO95/06058, EP 439 508, WO 97/03106, WO 96/21469, WO 95/13312, EP 921 131,WO 98/05363, EP 809 996, WO 96/41813, WO 96/07670, EP 605 963, EP 510356, EP 400 472, EP 183 503 and EP 154 316, which are incorporated byreference herein. Any of the PEG molecules described herein may be usedin any form, including but not limited to, single chain, branched chain,multiarm chain, single functional, bi-functional, multi-functional, orany combination thereof.

Enhancing Affinity for Serum Albumin

Various molecules can also be fused to the BPFIs of the invention tomodulate the half-life of BPFI in serum. In some embodiments, moleculesare linked or fused to BPFIs of the invention to enhance affinity forendogenous serum albumin in an animal.

For example, in some cases, a recombinant fusion of a BPFI and analbumin binding sequence is made. Exemplary albumin binding sequencesinclude, but are not limited to, the albumin binding domain fromstreptococcal protein G (see.e. g., Makrides et al., J Pharmacol. Exp.Ther. 277:534-542 (1996) and Sjolander et al., J, Immunol. Methods201:115-123 (1997)), or albumin-binding peptides such as those describedin, e.g., Dennis, et al., J. Biol. Chem. 277:35035-35043 (2002).

In other embodiments, the BPFIs of the present invention are acylatedwith fatty acids. In some cases, the fatty acids promote binding toserum albumin. See, e.g., Kurtzhals, et al., Biochem. J. 312:725-731(1995).

In other embodiments, the BPFIs of the invention are fused directly withserum albumin (including but not limited to, human serum albumin). Thoseof skill in the art will recognize that a wide variety of othermolecules can also be linked to BPFI in the present invention tomodulate binding to serum albumin or other serum components.

X Glycosylation of BPFI

The invention includes BPFIs incorporating one or more non-naturallyencoded amino acids bearing saccharide residues. The saccharide residuesmay be either natural (including but not limited to,N-acetylglucosamine) or non-natural (including but not limited to,3-fluorogalactose). The saccharides may be linked to the non-naturallyencoded amino acids either by an N- or O-linked glycosidic linkage(including but not limited to, N-acetylgalactose-L-serine) or anon-natural linkage (including but not limited to, an oxime or thecorresponding C- or S-linked glycoside).

The saccharide (including but not limited to, glycosyl) moieties can beadded to BPFIs either in vivo or in vitro. In some embodiments of theinvention, a BPFI comprising a carbonyl-containing non-naturally encodedamino acid is modified with a saccharide derivatized with an aminooxygroup to generate the corresponding glycosylated polypeptide linked viaan oxime linkage. Once attached to the non-naturally encoded amino acid,the saccharide may be further elaborated by treatment withglycosyltransferases and other enzymes to generate an oligosaccharidebound to the BPFI. See, e.g., H. Liu, et al. J. Am. Chem. Soc. 125:1702-1703 (2003).

In some embodiments of the invention, a BPFI comprising acarbonyl-containing non-naturally encoded amino acid is modifieddirectly with a glycan with defined structure prepared as an aminooxyderivative. One skilled in the art will recognize that otherfunctionalities, including azide, alkyne, hydrazide, hydrazine, andsemicarbazide, can be used to link the saccharide to the non-naturallyencoded amino acid.

In some embodiments of the invention, a BPFI comprising an azide oralkynyl-containing non-naturally encoded amino acid can then be modifiedby, including but not limited to, a Huisgen [3+2] cycloaddition reactionwith, including but not limited to, alkynyl or azide derivatives,respectively. This method allows for proteins to be modified withextremely high selectivity.

XI. BPFI Containing Dimers and Multimers

The present invention also provides for BPFI combinations such ashomodimers, heterodimers, homomultimers, or heteromultimers (i.e.,trimers, tetramers, etc.) where a particular BPFI containing one or morenon-naturally encoded amino acids is bound to another BPFI, analog, orvariant thereof or any other polypeptide that is a non-BPFI peptide orvariant thereof, either directly to the polypeptide backbone or via alinker. Due to its increased molecular weight compared to monomers, theBPFI dimer or multimer conjugates may exhibit new or desirableproperties, including but not limited to different pharmacological,pharmacokinetic, pharmacodynamic, modulated therapeutic half-life, ormodulated plasma half-life relative to the monomeric BPFI. In someembodiments, the conjugates or fusions of the invention will modulatethe interaction of the BPFI with its receptor or binding partner. Inother embodiments, the BPFI conjugates, fusions, dimers or multimers ofthe present invention will act as a receptor antagonist, agonist, superagonist, or modulator.

In some embodiments, one or more of the BPFIs present in a BPFIcontaining dimer or multimer comprises a non-naturally encoded aminoacid liked to a water soluble polymer that is present in the receptorbinding region or region for binding to a binding partner. In someembodiments, the BPFIs are linked directly, including but not limitedto, via an Asn-Lys amide linkage or Cys-Cys disulfide linkage. In someembodiments, the linked BPFI will comprise different non-naturallyencoded amino acids to facilitate conjugation, fusion, dimerization, ormultimerization including but not limited to, an alkyne in onenon-naturally encoded amino acid of a first BPFI and an azide in asecond non-naturally encoded amino acid of a second BPFI will beconjugated via a Huisgen [3+2] cycloaddition. Alternatively, a firstBPFI, and/or the linked BPFI comprising a ketone-containingnon-naturally encoded amino acid can be conjugated to a second BPFIcomprising a hydroxylamine-containing non-naturally encoded amino acidand the polypeptides are reacted via formation of the correspondingoxime.

Alternatively, the two BPFIs are linked via a linker. Any hetero- orhomo-bifunctional linker can be used to link the two BPFIs, which canhave the same or different primary sequence. In some cases, the linkerused to tether the BPFIs together can be a bifunctional PEG reagent. Thelinker may have a wide range of molecular weight or molecular length.Larger or smaller molecular weight linkers may be used to provide adesired spatial relationship or conformation between the BPFI and thelinked entity, or between the BPFI and its binding partner, or betweenthe linked entity and its binding partner, if any. Linkers having longeror shorter molecular length may also be used to provide a desired spaceor flexibility between the BPFI and the linked entity, or between theBPFI and its binding partner, or between the linked entity and itsbinding partner, if any. Similarly, a linker having a particular shapeor conformation may be utilized to impart a particular shape orconformation to the BPFI or the linked entity, either before or afterthe BPFI reaches its target. This optimization of the spatialrelationship between the BPFI and the linked entity and the bindingpartner may provide new, modulated, or desired properties to themolecule.

In some embodiments, the invention provides water-soluble bifunctionallinkers that have a dumbbell structure that includes: a) an azide, analkyne, a hydrazine, a hydrazide, a hydroxylamine, or acarbonyl-containing moiety on at least a first end of a polymerbackbone; and b) at least a second functional group on a second end ofthe polymer backbone. The second functional group can be the same ordifferent as the first functional group. The second functional group, insome embodiments, is not reactive with the first functional group. Theinvention provides, in some embodiments, water-soluble compounds thatcomprise at least one arm of a branched molecular structure. Forexample, the branched molecular structure can be dendritic.

In some embodiments, the invention provides multimers comprising one ormore GH supergene family member, such as BPFI, formed by reactions withwater soluble activated polymers that have the structure:

R—(CH₂CH₂O)_(n)—O—(CH₂)_(m)—X

wherein n is from about 5 to 3,000, m is 2-10, X can be an azide, analkyne, a hydrazine, a hydrazide, an aminooxy group, a hydroxylamine, aacetyl, or carbonyl-containing moiety, and R is a capping group, afunctional group, or a leaving group that can be the same or differentas X. R can be, for example, a functional group selected from the groupconsisting of hydroxyl, protected hydroxyl, alkoxyl,N-hydroxysuccinimidyl ester, 1-benzotriazolyl ester,N-hydroxysuccinimidyl carbonate, 1-benzotriazolyl carbonate, acetal,aldehyde, aldehyde hydrates, alkenyl, acrylate, methacrylate,acrylamide, active sulfone, amine, aminooxy, protected amine, hydrazide,protected hydrazide, protected thiol, carboxylic acid, protectedcarboxylic acid, isocyanate, isothiocyanate, maleimide, vinylsulfone,dithiopyridine, vinylpyridine, iodoacetamide, epoxide, glyoxals, diones,mesylates, tosylates, and tresylate, alkene, and ketone.

XII. Measurement of BPFI Activity and Affinity of BPFI for the BPFIReceptor or Binding Partner

BPFI activity can be determined using standard in vitro or in vivoassays.

A number of assays may be used to monitor the activity of BPFIs of theinvention. Antiviral activity assays may be performed as described inBudge et al. J. or Virology 2004 May; 78(10); 5015-5022, including butnot limited to, antigen reduction assays, inhibition of viral attachmentassays, and post-attachment inhibition of viral infectivity assays. Invitro assays that test the BPFI's ability to inhibit syncytia formationmay be used as described in Pastey et al. Nature Medicine 2000;6(1):35-40. Additional assays include cell-to-cell fusion assays,competitive ELISA assays, and animal models for RSV may also be used tomeasure BPFI activity, as described in Pastey et al. Nature Medicine2000 January; 6(1):35-40. Additional assays include, but are not limitedto, a cell based assay that measures the induction of cytopathologiceffect (CPE) on cells infected with RSV, infection assays utilizing aRSV reporter virus, and assays testing the effect of peptides on the Aand B strains of RSV. Alternatively, a number of other assays includingbut not limited to, other assays measuring antiviral activity, includingbut not limited to, assays measuring viral entry or viral fusion, knownto one skilled in the art may be used to monitor the activity of BPFI ofthe invention. Modifications to these assays to test combination therapywith another antiviral agent are also known to one skilled in the art.

Also, standard methods which are well-known to those of skill in the artmay be utilized for assaying non-retroviral activity. See, for example,Pringle et al. (Pringle, C. R. et al., 1985, J. Medical Virology17:377-386) for a discussion of respiratory syncytial virus andparainfluenza virus activity assay techniques. Further, see, forexample, “Zinsser Microbiology”, 1988, Joklik, W. K. et al., eds.,Appleton & Lange, Norwalk, Conn., 19th ed., for a general review of suchtechniques. These references are incorporated by reference herein in itsentirety. Animal studies may be performed with BPFI of the invention.Such studies include, but are not limited to, toxicity studies.

Regardless of which methods are used to create the BPFI analogs, theanalogs are subject to assays for biological activity. In general, thetest for biological activity should provide analysis for the desiredresult, such as increase or decrease in biological activity (as comparedto non-altered BPFI), different biological activity (as compared tonon-altered BPFI), receptor or binding partner affinity analysis,conformational or structural changes of the BPFI itself or bindingpartner (as compared to the non-altered BPFI), or serum half-lifeanalysis.

The above compilation of references for assay methodologies is notexhaustive, and those skilled in the art will recognize other assaysuseful for testing for the desired end result.

XIII. Measurement of Potency, Functional In Vivo Half-Life, andPharmacokinetic Parameters

An important aspect of the invention is the prolonged biologicalhalf-life that is obtained by construction of the BPFI with or withoutconjugation of the polypeptide to a water soluble polymer moiety. Therapid decrease of BPFI serum concentrations has made it important toevaluate biological responses to treatment with conjugated andnon-conjugated BPFI and variants thereof. Preferably, the conjugated andnon-conjugated BPFI and variants thereof of the present invention haveprolonged serum half-lives also after i.v. administration, making itpossible to measure by, e.g. ELISA method or by a primary screeningassay. Measurement of in vivo biological half-life may be carried out asdescribed herein.

Pharmacokinetic parameters for a BPFI comprising a non-naturally encodedamino acid can be evaluated in normal Sprague-Dawley male rats (N=5animals per treatment group). Animals will receive either a single doseof 25 ug/rat iv or 50 ug/rat sc, and approximately 5-7 blood sampleswill be taken according to a pre-defined time course, generally coveringabout 6 hours for a BPFI comprising a non-naturally encoded amino acidnot conjugated to a water soluble polymer and about 4 days for a BPFIcomprising a non-naturally encoded amino acid and conjugated to a watersoluble polymer.

A BPFI's ability to inhibit RSV entry into cells or viral fusion can beassessed in vitro (e.g., in a syncytium assay, an infectivity assay) orin vivo (e.g. in an appropriate animal model or in humans).

The specific activity of BPFIs in accordance with this invention can bedetermined by various assays known in the art. The biological activityof the BPFI muteins, or fragments thereof, obtained and purified inaccordance with this invention can be tested by methods described orreferenced herein or known to those skilled in the art.

XIV. Administration and Pharmaceutical Compositions

The polypeptides or proteins of the invention (including but not limitedto, BPFI, synthetases, proteins comprising one or more unnatural aminoacid, etc.) are optionally employed for therapeutic uses, including butnot limited to, in combination with a suitable pharmaceutical carrier.Such compositions, for example, comprise a therapeutically effectiveamount of the compound, and a pharmaceutically acceptable carrier orexcipient. Such a carrier or excipient includes, but is not limited to,saline, buffered saline, dextrose, water, glycerol, ethanol, and/orcombinations thereof. The formulation is made to suit the mode ofadministration. In general, methods of administering proteins are wellknown in the art and can be applied to administration of thepolypeptides of the invention.

Therapeutic compositions comprising one or more polypeptide of theinvention are optionally tested in one or more appropriate in vitroand/or in vivo animal models of disease, to confirm efficacy, tissuemetabolism, and to estimate dosages, according to methods well known inthe art. In particular, dosages can be initially determined by activity,stability or other suitable measures of unnatural herein to naturalamino acid homologues (including but not limited to, comparison of aBPFI modified to include one or more unnatural amino acids to a naturalamino acid BPFI), i.e., in a relevant assay.

Administration is by any of the routes normally used for introducing amolecule into ultimate contact with blood or tissue cells. The unnaturalamino acid polypeptides of the invention are administered in anysuitable manner, optionally with one or more pharmaceutically acceptablecarriers. Suitable methods of administering such polypeptides in thecontext of the present invention to a patient are available, and,although more than one route can be used to administer a particularcomposition, a particular route can often provide a more immediate andmore effective action or reaction than another route.

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of pharmaceutical compositions of thepresent invention.

Polypeptide compositions can be administered by a number of routesincluding, but not limited to oral, intravenous, intraperitoneal,intramuscular, transdermal, subcutaneous, topical, sublingual, or rectalmeans. Compositions comprising non-natural amino acid polypeptides,modified or unmodified, can also be administered via liposomes. Suchadministration routes and appropriate formulations are generally knownto those of skill in the art.

The BPFI comprising a non-natural amino acid, alone or in combinationwith other suitable components, can also be made into aerosolformulations (i.e., they can be “nebulized”) to be administered viainhalation. Aerosol formulations can be placed into pressurizedacceptable propellants, such as dichlorodifluoromethane, propane,nitrogen, and the like.

Formulations suitable for parenteral administration, such as, forexample, by intraarticular (in the joints), intravenous, intramuscular,intradermal, intraperitoneal, and subcutaneous routes, include aqueousand non-aqueous, isotonic sterile injection solutions, which can containantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The formulations of BPFI can be presented in unit-dose or multi-dosesealed containers, such as ampules and vials.

Parenteral administration and intravenous administration are preferredmethods of administration. In particular, the routes of administrationalready in use for natural amino acid homologue therapeutics (includingbut not limited to, those typically used for GLP-1, DP-178, PYY, EPO,GH, G-CSF, GM-CSF, IFNs, interleukins, antibodies, and/or any otherpharmaceutically delivered polypeptide or protein), along withformulations in current use, provide preferred routes of administrationand formulation for the polypeptides of the invention.

The dose administered to a patient, in the context of the presentinvention, is sufficient to have a beneficial therapeutic response inthe patient over time, or, including but not limited to, to inhibitinfection by a pathogen, or other appropriate activity, depending on theapplication. The dose is determined by the efficacy of the particularvector, or formulation, and the activity, stability or serum half-lifeof the unnatural amino acid polypeptide employed and the condition ofthe patient, as well as the body weight or surface area of the patientto be treated. The size of the dose is also determined by the existence,nature, and extent of any adverse side-effects that accompany theadministration of a particular vector, formulation, or the like in aparticular patient.

In determining the effective amount of the vector or formulation to beadministered in the treatment or prophylaxis of disease (including butnot limited to, cancers, inherited diseases, diabetes, AIDS, or thelike), the physician evaluates circulating plasma levels, formulationtoxicities, progression of the disease, and/or where relevant, theproduction of anti-unnatural amino acid polypeptide antibodies.

The dose administered, for example, to a 70 kilogram patient, istypically in the range equivalent to dosages of currently-usedtherapeutic proteins, adjusted for the altered activity or serumhalf-life of the relevant composition. The vectors of this invention cansupplement treatment conditions by any known conventional therapy,including antibody administration, vaccine administration,administration of cytotoxic agents, natural amino acid polypeptides,nucleic acids, nucleotide analogues, biologic response modifiers, andthe like.

For administration, formulations of the present invention areadministered at a rate determined by the LD-50 or ED-50 of the relevantformulation, and/or observation of any side-effects of the unnaturalamino acids at various concentrations, including but not limited to, asapplied to the mass and overall health of the patient. Administrationcan be accomplished via single or divided doses.

If a patient undergoing infusion of a formulation develops fevers,chills, or muscle aches, he/she receives the appropriate dose ofaspirin, ibuprofen, acetaminophen or other pain/fever controlling drug.Patients who experience reactions to the infusion such as fever, muscleaches, and chills are premedicated 30 minutes prior to the futureinfusions with either aspirin, acetaminophen, or, including but notlimited to, diphenhydramine. Meperidine is used for more severe chillsand muscle aches that do not quickly respond to antipyretics andantihistamines. Cell infusion is slowed or discontinued depending uponthe severity of the reaction.

Human BPFIs of the invention can be administered directly to a mammaliansubject. Administration is by any of the routes normally used forintroducing BPFI to a subject. The BPFI compositions according toembodiments of the present invention include those suitable for oral,rectal, topical, inhalation (including but not limited to, via anaerosol), buccal (including but not limited to, sub-lingual), vaginal,parenteral (including but not limited to, subcutaneous, intramuscular,intradermal, intraarticular, intrapleural, intraperitoneal,inracerebral, intraarterial, or intravenous), topical (i.e., both skinand mucosal surfaces, including airway surfaces) and transdermaladministration, although the most suitable route in any given case willdepend on the nature and severity of the condition being treated.Administration can be either local or systemic. The formulations ofcompounds can be presented in unit-dose or multi-dose sealed containers,such as ampoules and vials. BPFIs of the invention can be prepared in amixture in a unit dosage injectable form (including but not limited to,solution, suspension, or emulsion) with a pharmaceutically acceptablecarrier. BPFIs of the invention can also be administered by continuousinfusion (using, including but not limited to, minipumps such as osmoticpumps), single bolus or slow-release depot formulations.

Formulations suitable for administration include aqueous and non-aqueoussolutions, isotonic sterile solutions, which can contain antioxidants,buffers, bacteriostats, and solutes that render the formulationisotonic, and aqueous and non-aqueous sterile suspensions that caninclude suspending agents, solubilizers, thickening agents, stabilizers,and preservatives. Solutions and suspensions can be prepared fromsterile powders, granules, and tablets of the kind previously described.

The pharmaceutical compositions of the invention may comprise apharmaceutically acceptable carrier. Pharmaceutically acceptablecarriers are determined in part by the particular composition beingadministered, as well as by the particular method used to administer thecomposition. Accordingly, there is a wide variety of suitableformulations of pharmaceutical compositions (including optionalpharmaceutically acceptable carriers, excipients, or stabilizers) of thepresent invention (see, e.g., Remington's Pharmaceutical Sciences,17^(th) ed. 1985)).

Suitable carriers include buffers containing phosphate, borate, HEPES,citrate, and other organic acids; antioxidants including ascorbic acid;low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates, including glucose, mannose, ordextrins; chelating agents such as EDTA; divalent metal ions such aszinc, cobalt, or copper; sugar alcohols such as mannitol or sorbitol;salt-forming counter ions such as sodium; and/or nonionic surfactantssuch as Tween™, Pluronics™, or PEG.

BPFIs of the invention, including those linked to water soluble polymerssuch as PEG can also be administered by or as part of sustained-releasesystems. Sustained-release compositions include, including but notlimited to, semi-permeable polymer matrices in the form of shapedarticles, including but not limited to, films, or microcapsules.Sustained-release matrices include from biocompatible materials such aspoly(2-hydroxyethyl methacrylate) (Langer et al., J. Biomed. Mater.Res., 15: 267-277 (1981); Langer, Chem. Tech., 12: 98-105 (1982),ethylene vinyl acetate (Langer et al., supra) orpoly-D-(−)-3-hydroxybutyric acid (EP 133,988), polylactides (polylacticacid) (U.S. Pat. No. 3,773,919; EP 58,481), polyglycolide (polymer ofglycolic acid), polylactide co-glycolide (copolymers of lactic acid andglycolic acid) polyanhydrides, copolymers of L-glutamic acid andgamma-ethyl-L-glutamate (Sidman et al., Biopolymers, 22, 547-556 (1983),poly(ortho)esters, polypeptides, hyaluronic acid, collagen, chondroitinsulfate, carboxylic acids, fatty acids, phospholipids, polysaccharides,nucleic acids, polyamino acids, amino acids such as phenylalanine,tyrosine, isoleucine, polynucleotides, polyvinyl propylene,polyvinylpyrrolidone and silicone. Sustained-release compositions alsoinclude a liposomally entrapped compound. Liposomes containing thecompound are prepared by methods known per se: DE 3,218,121; Eppstein etal., Proc. Natl. Acad. Sci. U.S.A., 82: 3688-3692 (1985); Hwang et al.,Proc. Natl. Acad. Sci. U.S.A., 77: 4030-4034 (1980); EP 52,322; EP36,676; EP 88,046; EP 143,949; EP 142,641; Japanese Pat. Appln.83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324. Allreferences and patents cited are incorporated by reference herein.

Liposomally entrapped BPFIs can be prepared by methods described in,e.g., DE 3,218,121; Eppstein et al., Proc. Natl. Acad. Sci. U.S.A., 82:3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci. U.S.A., 77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP142,641; Japanese Pat. Appln. 83-118008; U.S. Pat. Nos. 4,485,045 and4,544,545; and EP 102,324. Composition and size of liposomes are wellknown or able to be readily determined empirically by one skilled in theart. Some examples of liposomes as described in, e.g., Park J W, et al.,Proc. Natl. Acad. Sci. USA 92:1327-1331 (1995); Lasic D andPapahadjopoulos D (eds): MEDICAL APPLICATIONS OF LIPOSOMES (1998);Drummond D C, et al., Liposomal drug delivery systems for cancertherapy, in Teicher B (ed): CANCER DRUG DISCOVERY AND DEVELOPMENT(2002); Park J W, et al., Clin. Cancer Res. 8:1172-1181 (2002); NielsenU B, et al., Biochim. Biophys. Acta 1591(1-3):109-118 (2002); Mamot C,et al., Cancer Res. 63: 3154-3161 (2003). All references and patentscited are incorporated by reference herein.

The dose administered to a patient in the context of the presentinvention should be sufficient to cause a beneficial response in thesubject over time. Generally, the total pharmaceutically effectiveamount of the BPFI of the present invention administered parenterallyper dose is in the range of about 0.01 μg/kg/day to about 100 μg/kg, orabout 0.05 mg/kg to about 1 mg/kg, of patient body weight, although thisis subject to therapeutic discretion. The frequency of dosing is alsosubject to therapeutic discretion, and may be more frequent or lessfrequent than the commercially available BPFI products approved for usein humans. Generally, a PEGylated BPFI of the invention can beadministered by any of the routes of administration described above.

XV. Therapeutic Uses of BPFIs of the Invention

The BPFIs of the invention are useful for treating a wide range ofdisorders.

Administration of the BPFI products of the present invention results inany of the activities demonstrated by other BPFI preparations in humans.The pharmaceutical compositions containing the BPFI products may beformulated at a strength effective for administration by various meansto a human patient experiencing disorders that may be affected by BPFIagonists or antagonists, either alone or as part of a condition ordisease. Average quantities of the BPFI product may vary and inparticular should be based upon the recommendations and prescription ofa qualified physician. The exact amount of BPFI is a matter ofpreference subject to such factors as the exact type of condition beingtreated, the condition of the patient being treated, as well as theother ingredients in the composition. The invention also provides foradministration of a therapeutically effective amount of another activeagent. The amount to be given may be readily determined by one skilledin the art based upon therapy with BPFI.

Therapeutic uses of BPFI include, but are not limited to, treating RSVinfection, inhibiting RSV entry, inhibiting entry of other envelopedviruses including but not limited to HIV. BPFIs of the inventionpreferably exhibit antiviral activity. BPFI may be used for prophylaxisagainst RSV. As such, the peptides may be used as inhibitors of humanand non-human viral and retroviral, especially HIV, transmission touninfected cells. The human retroviruses whose transmission may beinhibited by the peptides of the invention include, but are not limitedto all strains of HIV-1 and HIV-2 and the human T-lymphocyte viruses(HTLV-I and II). The non-human retroviruses whose transmission may beinhibited by the peptides of the invention include, but are not limitedto bovine leukosis virus, feline sarcoma and leukemia viruses, simianimmunodeficiency, sarcoma and leukemia viruses, and sheep progresspneumonia viruses. Non retroviral viruses whose transmission may beinhibited by the peptides of the invention include, but are not limitedto human respiratory syncytial virus. The invention further encompassesthe treatment of the above non-retroviral viruses using the peptides incombination therapy with at least one other therapeutic, including butnot limited to, an antiviral agent.

Another example of a peptide is T-20 (DP-178) which is a peptidecorresponding to amino acids 638 to 673 of the HIV-1_(LAI) transmembraneprotein (TM) gp41, the carboxyl-terminal helical segment of theextracellular portion of gp41. The extracellular portion of gp41 hasanother α-helical region which is the amino-terminal proposed zipperdomain, DP-107. DP-107 exhibits potent antiviral activity by inhibitingviral fusion. It is a 38 amino acid peptide, corresponding to residues558 to 595 of the HIV-1_(LAI) transmembrane gp41 protein. Studies withDP-107 have proven both are non-toxic in in vitro studies and inanimals. U.S. Pat. No. 5,656,480, which is incorporated by referenceherein, describes DP-107 and its antiviral activity.

T-20 inhibits entry of HIV into cells by acting as a viral fusioninhibitor. The fusion process of HIV is well characterized. HIV binds toCD4 receptor via gp120, and upon binding to its receptor, gp120 goesthrough a series of conformational changes that allows it to bind to itscoreceptors, CCR5 or CXCR4. After binding to both receptor andcoreceptor, gp120 exposes gp41 to begin the fusion process. gp41 has tworegions named heptad repeat 1 and 2 (HR1 and 2). The extracellulardomain identified as HR1 is an α-helical region which is theamino-terminal of a proposed zipper domain. HR1 comes together with HR2of gp41 to form a hairpin. The structure that it is formed is a 6-helixbundle that places the HIV envelope in the proximity of the cellularmembrane causing fusion between the two menbranes. T-20 prevents theconformational changes necessary for viral fusion by binding the firstheptad-repeat (HR1) of the gp41 transmembrane glycoprotein. Thus, theformation of the 6-helix bundle is blocked by T-20's binding to the HR1region of gp41. The DP107 and DP178 domains (i.e., the HR1 and HR2domains) of the HIV gp41 protein non-covalently complex with each other,and their interaction is required for the normal infectivity of thevirus. Compounds that disrupt the interaction between DP107 and DP178,and/or between DP107-like and DPI 78-like peptides are antifusogenic,including antiviral.

DP-178 acts as a potent inhibitor of HIV-1 mediated CD-4⁺ cell-cellfusion (i.e., syncytial formation) and infection of CD-4⁺ cells bycell-free virus. Such anti-retroviral activity includes, but is notlimited to, the inhibition of HIV transmission to uninfected CD-4⁺cells. DP-178 act at low concentrations, and it has been proven that itis non-toxic in in vitro studies and in animals. The amino acidconservation within the DP-178-corresponding regions of HIV-1 and HIV-2has been described.

Potential uses for DP-178 peptides are described in U.S. Pat. Nos.5,464,933 and 6,133,418, as well as U.S. Pat. Nos. 6,750,008 and6,824,783, all of which are incorporated by reference herein, for use ininhibition of fusion events associated with HIV transmission.

Portions, homologs, and analogs of DP178 and DP-107 as well asmodulators of DP178/DP107, DP178-like/DP107-like or HR1/HR2 interactionshave been investigated that show antiviral activity, and/or showanti-membrane fusion capability, or an ability to modulate intracellularprocesses involving coiled-coil peptide structures in retroviruses otherthan HIV-1 and nonretroviral viruses. Viruses in such studies include,simian immunodeficiency virus (U.S. Pat. No. 6,017,536), respiratorysynctial virus (U.S. Pat. Nos. 6,228,983; 6,440,656; 6,479,055;6,623,741), Epstein-Barr virus (U.S. Pat. Nos. 6,093,794; 6,518,013),parainfluenza virus (U.S. Pat. No. 6,333,395), influenza virus (U.S.Pat. Nos. 6,068,973; 6,060,065), and measles virus (U.S. Pat. No.6,013,263). All of which are incorporated by reference herein.

A commercially available form of DP-178 is Fuzeon® (enfuvirtide, RocheLaboratories Inc. and Trimeris, Inc.). Fuzeon® has an acetylated Nterminus and a carboxamide as the C-terminus, and is described by thefollowing primary amino acid sequence:CH₃CO-YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF-NH₂. It is used incombination with other antivirals in HIV-1 patients that show HIV-1replication despite ongoing antiretroviral therapy.

U.S. Pat. Nos. 5,464,933 and 6,824,783, which are incorporated byreference herein, describes DP-178, DP-178 fragments, analogs, andhomologs, including, but not limited to, molecules with amino andcarboxy terminal truncations, substitutions, insertions, deletions,additions, or macromolecular carrier groups as well as DP-178 moleculeswith chemical groups such as hydrophobic groups present at their aminoand/or carboxy termini. Additional variants, include but are not limitedto, those described in U.S. Pat. No. 6,830,893 and the derivatives ofDP-178 disclosed in U.S. Pat. No. 6,861,059. A set of T-20 hybridpolypeptides are described in U.S. Pat. Nos. 6,656,906, 6,562,787,6,348,568 and 6,258,782, and a DP-178-toxin fusion is described in U.S.Pat. No. 6,627,197.

HAART (Highly Active Anti-Retroviral Therapy) is the standard of therapyfor HIV which combines drugs from a few classes of antiretroviral agentsto reduce viral loads. U.S. Pat. No. 6,861,059, which is incorporated byreference herein, discloses methods of treating HIV-1 infection orinhibiting HIV-1 replication employing DP-178 or DP-107 or derivativesthereof, in combination with at least one other antiviral therapeuticagent such as a reverse transcriptase inhibitor (e.g. AZT, ddI, ddC,ddA, d4T, 3TC, or other dideoxynucleotides or dideoxyfluoronucleosides)or an inhibitor of protease (e.g. indinavir; ritonavir). Otherantivirals include cytokines (e.g., rIFNα, rIFNβ, rIFNγ), inhibitors ofviral mRNA capping (e.g. ribavirin), inhibitors of HIV protease (e.g.ABT-538 and MK-639), amphotericin B as a lipid-binding molecule withanti-HIV activity, and castanospermine as an inhibitor of glycoproteinprocessing. Potential combination therapies of other anti-viral agents,including but not limited to, reverse transcriptase inhibitors,integrase inhibitors, protease inhibitors, cytokine antagonists, andchemokine receptor modulators with T-20 are described in a number ofreferences including U.S. Pat. Nos. 6,855,724; 6,844,340; 6,841,558;6,833,457; 6,825,210; 6,811,780; 6,809,109; 6,806,265; 6,768,007;6,750,230; 6,706,706; 6,696,494; 6,673,821; 6,673,791; 6,667,314;6,642,237; 6,599,911; 6,596,729; 6,593,346; 6,589,962; 6,586,430;6,541,515; 6,538,002; 6,531,484; 6,511,994; 6,506,777; 6,500,844;6,498,161; 6,472,410; 6,432,981; 6,410,726; 6,399,619; 6,395,743;6,358,979; 6,265,434; 6,248,755; 6,245,806; and 6,172,110.

Potential delivery systems for DP-178 include, but are not limited tothose described in U.S. Pat. Nos. 6,844,324 and 6,706,892. In addition,a process for producing T-20 in inclusion bodies was described in U.S.Pat. No. 6,858,410.

T20/DP178, T21/DP107, and fragments thereof have also been found tointeract with N-formyl peptide receptor (FPR members). T-20 activatesthe N-formyl peptide receptor present in human phagocytes (Su et al.(1999) Blood 93(11):3885-3892) and is a chemoattractant and activator ofmonocytes and neutrophils (see U.S. Pat. No. 6,830,893). The FPR classreceptors are G-protein-coupled, STM receptors that bind thechemoattractant fMLP (N-formyl-methionyl-leucyl-phenylalanine) and areinvolved in monocyte chemotaxis and the induction of a host immuneresponse to a pathogen. The prototype FPR class receptor, FPR, bindsfMLP with high affinity and is activated by low concentrations of fMLP.The binding of FPR by fMLP induces a cascade of G protein-mediatedsignaling events leading to phagocytic cell adhesion, chemotaxis,release of oxygen intermediates, enhanced phagocytosis and bacterialkilling, as well as MAP kinase activation and gene transcription. (Krumpet al., J Biol Chem 272:937 (1997); Prossnitz et al., Pharmacol Ther74:73 (1997); Murphy, Annu. Rev. Immuno. 12: 593 (1994); and Murphy, TheN-formyl peptide chemotactic receptors, Chemoattractant ligands andtheir receptors. CRC Press, Boca Raton, p. 269 (1996)). Another FPRclass receptor is the highly homologous variant of FPR, named FPRL1(also referred to as FPRH2 and LXA4R). FPRL1 was originally cloned as anorphan receptor (Murphy et al., J. Biol. Chem., 267:7637-7643 (1992); Yeet al., Biochem. Biophys. Res. Commun., 184:582-589 (1992); Bao et al.,Genomics, 13:437-440 (1992); Gao, J. L. and P. M. Murphy, J. Biol.Chem., 268:25395-25401 (1993); and Nomura et al., Int. Immunol.,5:1239-1249 (1993)) but was subsequently found to mediate Ca²⁺mobilization in response to high concentrations of fMLP. (Ye et al.,Biochem. Biophys. Res. Commun., 184:582-589 (1992); and Gao, J. L. andP. M. Murphy, J. Biol. Chem., 268:25395-25401 (1993)).

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1

This example describes a few of the many potential sets of criteria forthe selection of preferred sites of incorporation of non-naturallyencoded amino acids into a BPFI. An optimal HR-C derived peptidecandidate is designed. Criteria such as peptide expression, stability,helical propensity, and anti-viral activity are assessed to identify anoptimal RSV peptide fusion inhibitor. Peptides optimal for helixformation upon a computer based analysis of the amino acid sequences arecloned into the expression vector. The peptides of variable lengths areproduced biosynthetically within the HR-C region of RSV F protein(position from 474 to 523). A fraction of the peptides are engineered tohave enhanced helical propensity using known helix favoring strategiesincluding helix end capping, tryptophan cages and salt bridge formation.The DNA coding region of each single peptide carrying specificrestriction sites are commercially synthesized for rapid cloning into anexpression vector.

The biosynthetically produced peptides are assayed for biologicalactivity. Peptides are analyzed by CD (circular dicroism) to determinewhich peptides display the best helical propensity.

HR-C analogue peptides are developed that retain RSV inhibitory potencyfollowing covalent attachment of polyethylene glycol side chains,utilizing a combination of site-directed placement and structuralmechanisms. Bifunctional heterodimeric peptides between a HR-C analogueand anionic peptides derived from RhoA are developed. Using HR-Cstructural data (Zhao et al. Proc Natl Acad Sci USA. 2000 Dec. 19;97(26):14172-7), PEG attachment positions in the peptide or peptides areselected based on solvent exposure and exposed helix faces. Mutants arealso constructed with amber codon substitutions at each position of theselected peptide coding sequence (up to 50 different mutants will begenerated) providing the potential to incorporate pAcF at each positionin the peptide(s). Suppression efficiency is assessed for amber codonsubstitution mutants by SDS-PAGE. Each of the positions is evaluated forsuppression efficiency. pAcF (para-acetyl phenylalanine) substitutedpeptides are produced biosynthetically and are PEGylated on pAcF using30 kDa PEG. Anti-viral activity of the RSV peptide analogues are testedin cell based assays. To further assess anti-viral activity andinhibition of syncytia formation, assays including but not limited tocell-cell fusion assay using primary human respiratory epithelium cellsare used. Binding of candidate peptide to RSV F protein is performed.Circular dichroism (CD) and differential scanning calorimetry (DSC) onthe pAcF substituted and PEGylated peptides are performed to determinetheir helix propensity and stability.

A heterodimeric or multimeric peptide molecule that is composed of aHR-C derived peptide and a small anionic peptide (including but notlimited to 15 mers to 19 mers) derived from the GTPase RhoA sequencethat could be multimerized or linked to the HR-C analogue peptides aredesigned. Bispecific peptides using the best candidate and a GTPase RhoAderived peptide are created. Synthetically produced 5 anionic RhoApeptides of variable length are tested in anti-viral assays. One or moreRhoA derived peptides are linked to one or more HR-C analogue peptidesthrough a linker connecting the unnatural amino acid pAcF. The linkageare varied between the two peptides to obtain the most biological activemolecule. This linkage (length of linker can be varied) results in theformation of bispecific peptides that are tested for antiviralactivities, including but not limited to, ability to block RSVattachment to the specific cell receptor and inhibit viral fusion to thecell membrane. This type of bispecific peptide construct may provideincreased anti-viral potency since two separate inhibition mechanismsare being utilized. Bispecific peptides are made and tested inanti-viral and cell fusion assays. FIG. 13 is a schematic of thepotential mechanism of action of a BPFI for RSV.

Example 2

This example details expression of BPFI including a non-naturallyencoded amino acid in E. coli.

An introduced translation system that comprises an orthogonal tRNA(O-tRNA) and an orthogonal aminoacyl tRNA synthetase (O-RS) is used toexpress BPFI containing a non-naturally encoded amino acid. The O-RSpreferentially aminoacylates the O-tRNA with a non-naturally encodedamino acid. In turn the translation system inserts the non-naturallyencoded amino acid into BPFI, in response to an encoded selector codon.

TABLE 2 O-RS and O-tRNA sequences. SEQ ID NO: 2 M. jannaschiiMtRNA_(CUA) ^(Tyr) tRNA SEQ ID NO: 3 HLAD03; an optimized ambersupressor tRNA tRNA SEQ ID NO: 4 HL325A; an optimized AGGA tRNAframeshift supressor tRNA SEQ ID NO: 5 Aminoacyl tRNA synthetase for theincorporation RS of p-azido-L-phenylalanine p-Az-PheRS(6) SEQ ID NO: 6Aminoacyl tRNA synthetase for the incorporation RS ofp-benzoyl-L-phenylalanine p-BpaRS(1) SEQ ID NO: 7 Aminoacyl tRNAsynthetase for the incorporation RS of propargyl-phenylalaninePropargyl-PheRS SEQ ID NO: 8 Aminoacyl tRNA synthetase for theincorporation RS of propargyl-phenylalanine Propargyl-PheRS SEQ ID NO: 9Aminoacyl tRNA synthetase for the incorporation RS ofpropargyl-phenylalanine Propargyl-PheRS SEQ ID NO: 10 Aminoacyl tRNAsynthetase for the incorporation RS of p-azido-phenylalaninep-Az-PheRS(1) SEQ ID NO: 11 Aminoacyl tRNA synthetase for theincorporation RS of p-azido-phenylalanine p-Az-PheRS(3) SEQ ID NO: 12Aminoacyl tRNA synthetase for the incorporation RS ofp-azido-phenylalanine p-Az-PheRS(4) SEQ ID NO: 13 Aminoacyl tRNAsynthetase for the incorporation RS of p-azido-phenylalaninep-Az-PheRS(2) SEQ ID NO: 14 Aminoacyl tRNA synthetase for theincorporation RS of p-acetyl-phenylalanine (LW1) SEQ ID NO: 15 AminoacyltRNA synthetase for the incorporation RS of p-acetyl-phenylalanine (LW5)SEQ ID NO: 16 Aminoacyl tRNA synthetase for the incorporation RS ofp-acetyl-phenylalanine (LW6) SEQ ID NO: 17 Aminoacyl tRNA synthetase forthe incorporation RS of p-azido-phenylalanine (AzPheRS-5) SEQ ID NO: 18Aminoacyl tRNA synthetase for the incorporation RS ofp-azido-phenylalanine (AzPheRS-6)The transformation of E. coli with plasmids containing the modified BPFIgene and the orthogonal aminoacyl tRNA synthetase/tRNA pair (specificfor the desired non-naturally encoded amino acid) allows thesite-specific incorporation of non-naturally encoded amino acid into theBPFI. The transformed E. coli, grown at 37° C. in media containingbetween 0.01-100 mM of the particular non-naturally encoded amino acid,expresses modified BPFI with high fidelity and efficiency.

Example 3

This example details introduction of a carbonyl-containing amino acidand subsequent reaction with an aminooxy-containing PEG.

This Example demonstrates a method for the generation of a BPFI thatincorporates a ketone-containing non-naturally encoded amino acid thatis subsequently reacted with an aminooxy-containing PEG of approximately5,000 MW. For example, each of the residues in a BPFI is separatelysubstituted with a non-naturally encoded amino acid having the followingstructure:

The sequences utilized for site-specific incorporation ofp-acetyl-phenylalanine into BPFI and SEQ ID NO: 2 (muttRNA, M jannaschiimtRNA_(CUA) ^(Tyr)), and 14, 15 or 16 (TyrRS LW1, 5, or 6) described inExample 2 above.

Once modified, the BPFI variant comprising the carbonyl-containing aminoacid is reacted with an aminooxy-containing PEG derivative of the form:

R-PEG(N)—O—(CH₂)_(n)—O—NH₂

where R is methyl, n is 3 and N is approximately 5,000 MW. The purifiedBPFI containing p-acetylphenylalanine dissolved at 10 mg/mL in 25 mM MES(Sigma Chemical, St. Louis, Mo.) pH 6.0, 25 mM Hepes (Sigma Chemical,St. Louis, Mo.) pH 7.0, or in 10 mM Sodium Acetate (Sigma Chemical, St.Louis, Mo.) pH 4.5, is reacted with a 10 to 100-fold excess ofaminooxy-containing PEG, and then stirred for 10-16 hours at roomtemperature (Jencks, W. J. Am. Chem. Soc. 1959, 81, pp 475). ThePEG-BPFI is then diluted into appropriate buffer for immediatepurification and analysis.

Example 4

Conjugation with a PEG consisting of a hydroxylamine group linked to thePEG via an amide linkage.

A PEG reagent having the following structure is coupled to aketone-containing non-naturally encoded amino acid using the proceduredescribed in Example 3:

R-PEG(N)—O—(CH₂)₂—NH—C(O)(CH₂)_(n)—O—NH₂

where R=methyl, n=4 and N is approximately 20,000 MW. The reaction,purification, and analysis conditions are as described in Example 3.

Example 5

This example details the introduction of two distinct non-naturallyencoded amino acids into a BPFI.

This example demonstrates a method for the generation of a BPFI thatincorporates non-naturally encoded amino acid comprising a ketonefunctionality at two positions, wherein X* represents a non-naturallyencoded amino acid. The BPFI is prepared as described in Examples 1 and2, except that the selector codon is introduced at two distinct siteswithin the nucleic acid.

Example 6

This example details conjugation of a BPFI to a hydrazide-containing PEGand subsequent in situ reduction.

A BPFI incorporating a carbonyl-containing amino acid is preparedaccording to the procedure described in Examples 2 and 3. Once modified,a hydrazide-containing PEG having the following structure is conjugatedto the BPFI:

R-PEG(N)—O—(CH₂)₂—NH—C(O)(CH₂)_(n)—X—NH—NH₂

where R=methyl, n=2 and N=10,000 MW and X is a carbonyl (CO)=group. Thepurified BPFI containing p-acetylphenylalanine is dissolved at between0.1-10 mg/mL in 25 mM MES (Sigma Chemical, St. Louis, Mo.) pH 6.0, 25 mMHepes (Sigma Chemical, St. Louis, Mo.) pH 7.0, or in 10 mM SodiumAcetate (Sigma Chemical, St. Louis, Mo.) pH 4.5, is reacted with a 1 to100-fold excess of hydrazide-containing PEG, and the correspondinghydrazone is reduced in situ by addition of stock 1M NaCNBH₃ (SigmaChemical, St. Louis, Mo.), dissolved in H₂O, to a final concentration of10-50 mM. Reactions are carried out in the dark at 4° C. to RT for 18-24hours. Reactions are stopped by addition of 1 M Tris (Sigma Chemical,St. Louis, Mo.) at about pH 7.6 to a final Tris concentration of 50 mMor diluted into appropriate buffer for immediate purification.

Example 7

This example details introduction of an alkyne-containing amino acidinto BPFI and derivatization with mPEG-azide.

Any of the residues of BPFI are each substituted with the followingnon-naturally encoded amino acid:

The sequences utilized for site-specific incorporation ofp-propargyl-tyrosine into BPFI, SEQ ID NO: 2 (m uttRNA, M. jannaschiimtRNA_(CUA) ^(Tyr)), and 7, 8 or 9 described in Example 2 above. TheBPFI containing the propargyl tyrosine is expressed in E. coli andpurified using the conditions described in Example 3.

The purified BPFI containing propargyl-tyrosine dissolved at between0.1-10 mg/mL in PB buffer (100 mM sodium phosphate, 0.15 M NaCl, pH=8)and a 10 to 1000-fold excess of an azide-containing PEG is added to thereaction mixture. A catalytic amount of CuSO₄ and Cu wire are then addedto the reaction mixture. After the mixture is incubated (including butnot limited to, about 4 hours at room temperature or 37° C., orovernight at 4° C.), H₂O is added and the mixture is filtered through adialysis membrane. The sample can be analyzed for the addition,including but not limited to, by similar procedures described in Example3.

In this Example, the PEG will have the following structure:

R-PEG(N)—O—(CH₂)₂—NH—C(O)(CH₂)_(n)—N₃

where R is methyl, n is 4 and N is 10,000 MW.

Example 8

This example details substitution of a large, hydrophobic amino acid ina BPFI with propargyl tyrosine.

A Phe, Trp or Tyr residue present within BPFI is substituted with thefollowing non-naturally encoded amino acid as described in Example 7:

Once modified, a PEG is attached to the BPFI variant comprising thealkyne-containing amino acid. The PEG will have the following structure:

Me-PEG(N)—O—(CH₂)₂—N₃

and coupling procedures would follow those in Example 7. This willgenerate a BPFI variant comprising a non-naturally encoded amino acidthat is approximately isosteric with one of the naturally-occurring,large hydrophobic amino acids and which is modified with a PEGderivative at a distinct site within the polypeptide.

Example 9

This example details generation of a BPFI homodimer, heterodimer,homomultimer, or heteromultimer separated by one or more PEG linkers.

The alkyne-containing BPFI variant produced in Example 7 is reacted witha bifunctional PEG derivative of the form:

N₃—(CH₂)_(n)—C(O)—NH—(CH₂)₂—O-PEG(N)—O—(CH₂)₂—NH—C(O)—(CH₂)_(n)—N₃

where n is 4 and the PEG has an average MW of approximately 5,000, togenerate the corresponding BPFI homodimer where the two BPFI moleculesare physically separated by PEG. In an analogous manner a BPFI may becoupled to one or more other polypeptides to form heterodimers,homomultimers, or heteromultimers. Coupling, purification, and analyseswill be performed as in Examples 7 and 3.

Example 10

This example details coupling of a saccharide moiety to BPFI.

One residue of BPFI is substituted with the non-naturally encoded aminoacid below, as described in Example 3.

Once modified, the BPFI variant comprising the carbonyl-containing aminoacid is reacted with a β-linked aminooxy analogue of N-acetylglucosamine(GlcNAc). The BPFI variant (10 mg/mL) and the aminooxy saccharide (21mM) are mixed in aqueous 100 mM sodium acetate buffer (pH 5.5) andincubated at 37° C. for 7 to 26 hours. A second saccharide is coupled tothe first enzymatically by incubating the saccharide-conjugated BPFI (5mg/mL) with UDP-galactose (16 mM) and β-1,4-galacytosyltransferase (0.4units/mL) in 150 mM HEPES buffer (pH 7.4) for 48 hours at ambienttemperature (Schanbacher et al. J. Biol. Chem. 1970, 245, 5057-5061).

Example 11

This example details generation of a PEGylated BPFI antagonist.

A residues of BPFI is substituted with the following non-naturallyencoded amino acid as described in Example 3.

Once modified, the BPFI comprising the carbonyl-containing amino acidwill be reacted with an aminooxy-containing PEG derivative of the form:

R-PEG(N)—O—(CH₂)_(n)—O—NH₂

where R is methyl, n is 4 and N is 20,000 MW to generate a BPFIantagonist comprising a non-naturally encoded amino acid that ismodified with a PEG derivative at a single site within the polypeptide.Coupling, purification, and analyses are performed as in Example 3.

Example 12 Generation of a BPFI Homodimer, Heterodimer, Homomultimer, orHeteromultimer in which the BPFI Molecules are Linked Directly

A BPFI variant comprising the alkyne-containing amino acid can bedirectly coupled to another BPFI variant comprising the azido-containingamino acid, each of which comprise non-naturally encoded amino acid. Inan analogous manner a BPFI may be coupled to one or more otherpolypeptides to form heterodimers, homomultimers, or heteromultimers.Coupling, purification, and analyses are performed as in Examples 3, 6,and 7.

Example 13

The polyalkylene glycol (P—OH) is reacted with the alkyl halide (A) toform the ether (B). In these compounds, n is an integer from one to nineand R′ can be a straight- or branched-chain, saturated or unsaturatedC1, to C20 alkyl or heteroalkyl group. R′ can also be a C3 to C7saturated or unsaturated cyclic alkyl or cyclic heteroalkyl, asubstituted or unsubstituted aryl or heteroaryl group, or a substitutedor unsubstituted alkaryl (the alkyl is a C1 to C20 saturated orunsaturated alkyl) or heteroalkaryl group. Typically, PEG-OH ispolyethylene glycol (PEG) or monomethoxy polyethylene glycol (mPEG)having a molecular weight of 800 to 40,000 Daltons (Da).

Example 14

mPEG-OH+Br—CH₂—C≡CH→mPEG-O—CH₂—C≡CH

mPEG-OH with a molecular weight of 20,000 Da (mPEG-OH 20 kDa; 2.0 g, 0.1mmol, Sunbio) was treated with NaH (12 mg, 0.5 mmol) in THF (35 mL). Asolution of propargyl bromide, dissolved as an 80% weight solution inxylene (0.56 mL, 5 mmol, 50 equiv., Aldrich), and a catalytic amount ofKI were then added to the solution and the resulting mixture was heatedto reflux for 2 hours. Water (1 mL) was then added and the solvent wasremoved under vacuum. To the residue was added CH₂Cl₂ (25 mL) and theorganic layer was separated, dried over anhydrous Na₂SO₄, and the volumewas reduced to approximately 2 mL. This CH₂Cl₂ solution was added todiethyl ether (150 mL) drop-wise. The resulting precipitate wascollected, washed with several portions of cold diethyl ether, and driedto afford propargyl-O-PEG.

Example 15

mPEG-OH+Br—(CH₂)₃—C≡CH→mPEG-O—(CH₂)₃—C≡CH

The mPEG-OH with a molecular weight of 20,000 Da (mPEG-OH 20 kDa; 2.0 g,0.1 mmol, Sunbio) was treated with NaH (12 mg, 0.5 mmol) in THF (35 mL).Fifty equivalents of 5-bromo-1-pentyne (0.53 mL, 5 mmol, Aldrich) and acatalytic amount of KI were then added to the mixture. The resultingmixture was heated to reflux for 16 hours. Water (1 mL) was then addedand the solvent was removed under vacuum. To the residue was addedCH₂Cl₂ (25 mL) and the organic layer was separated, dried over anhydrousNa₂SO₄, and the volume was reduced to approximately 2 mL. This CH₂Cl₂solution was added to diethyl ether (150 mL) drop-wise. The resultingprecipitate was collected, washed with several portions of cold diethylether, and dried to afford the corresponding alkyne. 5-chloro-1-pentynemay be used in a similar reaction.

Example 16

m-HOCH₂C₆H₄OH+NaOH+Br—CH₂—C≡CH→m-HOCH₂C₆H₄O—CH₂—C≡CH  (1)

m-HOCH₂C₆H₄O—CH₂—C≡CH+MsCl+N(Et)₃ →m-MsOCH₂C₆H₄O—CH₂—C≡CH  (2)

m-MsOCH₂C₆H₄O—CH₂—C≡CH+LiBr→m-Br—CH₂C₆H₄O—CH₂—C≡CH  (3)

mPEG-OH+m-Br—CH₂C₆H₄O—CH₂—C≡CH→mPEG-O—CH₂—C₆H₄O—CH₂—C≡CH  (4)

To a solution of 3-hydroxybenzylalcohol (2.4 g, 20 mmol) in THF (50 mL)and water (2.5 mL) was first added powdered sodium hydroxide (1.5 g,37.5 mmol) and then a solution of propargyl bromide, dissolved as an 80%weight solution in xylene (3.36 mL, 30 mmol). The reaction mixture washeated at reflux for 6 hours. To the mixture was added 10% citric acid(2.5 mL) and the solvent was removed under vacuum. The residue wasextracted with ethyl acetate (3×15 mL) and the combined organic layerswere washed with saturated NaCl solution (10 mL), dried over MgSO₄ andconcentrated to give the 3-propargyloxybenzyl alcohol.

Methanesulfonyl chloride (2.5 g, 15.7 mmol) and triethylamine (2.8 mL,20 mmol) were added to a solution of compound 3 (2.0 g, 11.0 mmol) inCH₂Cl₂ at 0° C. and the reaction was placed in the refrigerator for 16hours. A usual work-up afforded the mesylate as a pale yellow oil. Thisoil (2.4 g, 9.2 mmol) was dissolved in THF (20 mL) and LiBr (2.0 g, 23.0mmol) was added. The reaction mixture was heated to reflux for 1 hourand was then cooled to room temperature. To the mixture was added water(2.5 mL) and the solvent was removed under vacuum. The residue wasextracted with ethyl acetate (3×15 mL) and the combined organic layerswere washed with saturated NaCl solution (10 mL), dried over anhydrousNa₂SO₄, and concentrated to give the desired bromide.

mPEG-OH 20 kDa (1.0 g, 0.05 mmol, Sunbio) was dissolved in THF (20 mL)and the solution was cooled in an ice bath. NaH (6 mg, 0.25 mmol) wasadded with vigorous stirring over a period of several minutes followedby addition of the bromide obtained from above (2.55 g, 11.4 mmol) and acatalytic amount of KI. The cooling bath was removed and the resultingmixture was heated to reflux for 12 hours. Water (1.0 mL) was added tothe mixture and the solvent was removed under vacuum. To the residue wasadded CH₂Cl₂ (25 mL) and the organic layer was separated, dried overanhydrous Na₂SO₄, and the volume was reduced to approximately 2 mL.Dropwise addition to an ether solution (150 mL) resulted in a whiteprecipitate, which was collected to yield the PEG derivative.

Example 17

mPEG-NH₂+X—C(O)—(CH₂)_(n)—C≡CR′→mPEG-NH—C(O)—(CH₂)_(n)—C≡CR′

The terminal alkyne-containing poly(ethylene glycol) polymers can alsobe obtained by coupling a poly(ethylene glycol) polymer containing aterminal functional group to a reactive molecule containing the alkynefunctionality as shown above. n is between 1 and 10. R′ can be H or asmall alkyl group from C1 to C4.

Example 18

HO₂C—(CH₂)₂—C≡CH+NHS+DCC→NHSO—C(O)—(CH₂)₂—C≡CH  (1)

mPEG-NH₂+NHSO—C(O)—(CH₂)₂—C≡CH→mPEG-NH—C(O)—(CH₂)₂—C≡CH  (2)

4-pentynoic acid (2.943 g, 3.0 mmol) was dissolved in CH₂Cl₂ (25 mL).N-hydroxysuccinimide (3.80 g, 3.3 mmol) and DCC (4.66 g, 3.0 mmol) wereadded and the solution was stirred overnight at room temperature. Theresulting crude NHS ester 7 was used in the following reaction withoutfurther purification.

mPEG-NH₂ with a molecular weight of 5,000 Da (mPEG-NH₂, 1 g, Sunbio) wasdissolved in THF (50 mL) and the mixture was cooled to 4° C. NHS ester 7(400 mg, 0.4 mmol) was added portion-wise with vigorous stirring. Themixture was allowed to stir for 3 hours while warming to roomtemperature. Water (2 mL) was then added and the solvent was removedunder vacuum. To the residue was added CH₂Cl₂ (50 mL) and the organiclayer was separated, dried over anhydrous Na₂SO₄, and the volume wasreduced to approximately 2 mL. This CH₂Cl₂ solution was added to ether(150 mL) drop-wise. The resulting precipitate was collected and dried invacuo.

Example 19

This Example represents the preparation of the methane sulfonyl ester ofpoly(ethylene glycol), which can also be referred to as themethanesulfonate or mesylate of poly(ethylene glycol). The correspondingtosylate and the halides can be prepared by similar procedures.

mPEG-OH+CH₃SO₂Cl+N(Et)₃→mPEG-O—SO₂CH₃ →mPEG-N₃

The mPEG-OH (MW=3,400, 25 g, 10 mmol) in 150 mL of toluene wasazeotropically distilled for 2 hours under nitrogen and the solution wascooled to room temperature. 40 mL of dry CH₂Cl₂ and 2.1 mL of drytriethylamine (15 mmol) were added to the solution. The solution wascooled in an ice bath and 1.2 mL of distilled methanesulfonyl chloride(15 mmol) was added dropwise. The solution was stirred at roomtemperature under nitrogen overnight, and the reaction was quenched byadding 2 mL of absolute ethanol. The mixture was evaporated under vacuumto remove solvents, primarily those other than toluene, filtered,concentrated again under vacuum, and then precipitated into 100 mL ofdiethyl ether. The filtrate was washed with several portions of colddiethyl ether and dried in vacuo to afford the mesylate.

The mesylate (20 g, 8 mmol) was dissolved in 75 ml of THF and thesolution was cooled to 4° C. To the cooled solution was added sodiumazide (1.56 g, 24 mmol). The reaction was heated to reflux undernitrogen for 2 hours. The solvents were then evaporated and the residuediluted with CH₂Cl₂ (50 mL). The organic fraction was washed with NaClsolution and dried over anhydrous MgSO₄. The volume was reduced to 20 mland the product was precipitated by addition to 150 ml of cold dryether.

Example 20

N₃—C₆H₄—CO₂H→N₃—C₆H₄CH₂OH  (1)

N₃—C₆H₄CH₂OH→Br—CH₂—C₆H₄—N₃  (2)

mPEG-OH+Br—CH₂—C₆H₄—N₃ →mPEG-O—CH₂—C₆H₄—N₃  (3)

4-azidobenzyl alcohol can be produced using the method described in U.S.Pat. No. 5,998,595, which is incorporated by reference herein.Methanesulfonyl chloride (2.5 g, 15.7 mmol) and triethylamine (2.8 mL,20 mmol) were added to a solution of 4-azidobenzyl alcohol (1.75 g, 11.0mmol) in CH₂Cl₂ at 0° C. and the reaction was placed in the refrigeratorfor 16 hours. A usual work-up afforded the mesylate as a pale yellowoil. This oil (9.2 mmol) was dissolved in THF (20 mL) and LiBr (2.0 g,23.0 mmol) was added. The reaction mixture was heated to reflux for 1hour and was then cooled to room temperature. To the mixture was addedwater (2.5 mL) and the solvent was removed under vacuum. The residue wasextracted with ethyl acetate (3×15 mL) and the combined organic layerswere washed with saturated NaCl solution (10 mL), dried over anhydrousNa₂SO₄, and concentrated to give the desired bromide.

mPEG-OH 20 kDa (2.0 g, 0.1 mmol, Sunbio) was treated with NaH (12 mg,0.5 mmol) in THF (35 mL) and the bromide (3.32 g, 15 mmol) was added tothe mixture along with a catalytic amount of KI. The resulting mixturewas heated to reflux for 12 hours. Water (1.0 mL) was added to themixture and the solvent was removed under vacuum. To the residue wasadded CH₂Cl₂ (25 mL) and the organic layer was separated, dried overanhydrous Na₂SO₄, and the volume was reduced to approximately 2 mL.Dropwise addition to an ether solution (150 mL) resulted in aprecipitate, which was collected to yield mPEG-O—CH₂—C₆H₄—N₃.

Example 21

NH₂—PEG-O—CH₂CH₂CO₂H+N₃—CH₂CH₂CO₂—NHS→N₃—CH₂CH₂—C(O)NH-PEG-O—CH₂CH₂CO₂H

NH₂—PEG-O—CH₂CH₂CO₂H (MW 3,400 Da, 2.0 g) was dissolved in a saturatedaqueous solution of NaHCO₃ (10 mL) and the solution was cooled to 0° C.3-azido-1-N-hydroxysuccinimido propionate (5 equiv.) was added withvigorous stirring. After 3 hours, 20 mL of H₂O was added and the mixturewas stirred for an additional 45 minutes at room temperature. The pH wasadjusted to 3 with 0.5 N H₂SO₄ and NaCl was added to a concentration ofapproximately 15 wt %. The reaction mixture was extracted with CH₂Cl₂(100 mL×3), dried over Na₂SO₄ and concentrated. After precipitation withcold diethyl ether, the product was collected by filtration and driedunder vacuum to yield the omega-carboxy-azide PEG derivative.

Example 22

mPEG-OMs+HC≡CLi→mPEG-O—CH₂—CH₂—C≡C—H

To a solution of lithium acetylide (4 equiv.), prepared as known in theart and cooled to −78° C. in THF, is added dropwise a solution ofmPEG-OMs dissolved in THF with vigorous stirring. After 3 hours, thereaction is permitted to warm to room temperature and quenched with theaddition of 1 mL of butanol. 20 mL of H₂O is then added and the mixturewas stirred for an additional 45 minutes at room temperature. The pH wasadjusted to 3 with 0.5 N H₂SO₄ and NaCl was added to a concentration ofapproximately 15 wt %. The reaction mixture was extracted with CH₂Cl₂(100 mL×3), dried over Na₂SO₄ and concentrated. After precipitation withcold diethyl ether, the product was collected by filtration and driedunder vacuum to yield the 1-(but-3-ynyloxy)-methoxypolyethylene glycol(mPEG).

Example 23

The azide- and acetylene-containing amino acids were incorporatedsite-selectively into proteins using the methods described in L. Wang,et al., (2001), Science 292:498-500, J. W. Chin et al., Science301:964-7 (2003)), J. W. Chin et al., (2002), Journal of the AmericanChemical Society 124:9026-9027; J. W. Chin, & P. G. Schultz, (2002),Chem Bio Chem 3(11):1135-1137; J. W. Chin, et al., (2002), PNAS UnitedStates of America 99:11020-11024: and, L. Wang, & P. G. Schultz, (2002),Chem. Comm., 1:1-11. Once the amino acids were incorporated, thecycloaddition reaction was carried out with 0.01 mM protein in phosphatebuffer (PB), pH 8, in the presence of 2 mM PEG derivative, 1 mM CuSO₄,and ˜1 mg Cu-wire for 4 hours at 37° C.

Example 24

This example describes a few of the many potential sets of criteria forthe selection of preferred sites of incorporation of non-naturallyencoded amino acids into T-20.

This example demonstrates how preferred sites within the T-20polypeptide were selected for introduction of a non-naturally encodedamino acid. Sequence numbering used in this example is according to theamino acid sequence of T-20 (SEQ ID NO: 22) and TEX (SEQ ID NO: 24). TEXis an N-terminal extended polypeptide of T-20. Position numbers citedare based positions 638-673 of the T-20 peptide and 630-673 of the TEXpeptide, unless otherwise indicated. For example, position 639corresponds to the second amino acid in SEQ ID NO: 22. Those of skill inthe art will appreciate that amino acid positions corresponding topositions in SEQ ID NO: 22, can be readily identified in SEQ ID NO: 24,or any other T-20 molecule.

Modeling of the potential alpha helical structure of T-20 was performedbased on PDB 1DLB from W. Shu, J. Liu, H. Ji, L. Rading, S. Jiang, M.Lu, Helical Interactions in the HIV-1 gp41 Core Reveal Structural Basisfor the Inhibitory Activity of gp41 Peptides (Biochemistry 39:1634(2000). The following criteria were used to evaluate each position ofT-20 for the introduction of a non-naturally encoded amino acid: theresidue (a) should not be affected by alanine scanning mutagenesis, (b)should be surface exposed and exhibit minimal van der Waals or hydrogenbonding interactions with surrounding residues based on modeling, (c)may either be variable or non-essential without affecting activity inT-20 variants, (d) would result in conservative substitutions uponsubstitution with a non-naturally encoded amino acid and (e) could befound in either highly flexible regions or structurally rigid regions.In addition, further calculations were performed on the T-20 molecule,utilizing the Cx program (Pintar et al. (2002) Bioinformatics, 18, pp980) to evaluate the extent of protrusion for each protein atom from thepeptide. The results of the analysis of TEX amino acid positions fornon-natural amino acid incorporation is shown in FIG. 3.

In some embodiments, one or more non-naturally encoded amino acids areincorporated at any position in T-20 (including TEX), before the firstamino acid, an addition at the carboxy terminus, or any combinationthereof. In some embodiments, one or more non-naturally encoded aminoacids are incorporated at any position in T-20 (including TEX),including but not limited to, the residues as follows: W631, D632, I635,N636, N637, Y638, T639, S640, L641, L645, N651, or any combinationthereof.

Example 25 Cloning Strategy to Produce Biosynthetically T-20 and TEX

FIG. 9A shows a schematic of constructs that were designed toincorporate a non-naturally encoded amino acid into T-20 polypeptide andinto a polypeptide of T-20 extended at the N terminus (TEX). HIVproviral DNA was used to amplify the sequence encoding T-20 and TEX,including a methionine at the N terminus of the peptide product. Primersused to amplify T-20 sequence from HIV proviral DNA were F-T20 5′AAG CTTTGG ATG TAC ACA AGT TTA ATA CAC TCC3′ (SEQ ID NO: 26) and R-T20 5′GCGGAT CCC ATT AAA ACC AAT TCC ACA AAC TTG C3′ (SEQ ID NO: 27). Primersused to amplify TEX sequence from HIV proviral DNA were F-EXT20 5′CG AAGCTT TGG ATG GAG TGG GAT AGA GAA ATT AAC AAT TAC ACA AGT TTA ATA CACTCC3′ (SEQ ID NO: 28) and R-T20 (SEQ ID NO: 27). F-T20 AND F-EXT20contained a HindIII restriction site, and R-T20 contained a BamHI sitefor cloning.

T-20 and TEX sequences were cloned in frame into an expression vectorcontaining a TrpLE fusion partner (FP) and a nine histidine tag at the Nterminus of the fusion partner.

FIG. 10 shows a comparison of the wild-type T-20 and TEX sequences. Theextended version of the peptide T-20 (TEX) was generated using theprimers indicated above to amplify the corresponding DNA region of thegp41 heptad repeat 2 (HR2) (see FIG. 1). TEX is 8 amino acids longerthan T-20 at the N-terminus, providing a polypeptide that is 44 aminoacids in length. TEX corresponds to amino acids 630 to 673 of theHIV_(NL4-3) transmembrane protein (TM). T-20 corresponds to amino acids638 to 673 of the HIV_(NL4-3) transmembrane protein (TM). FIG. 4 showsthe production of TEX mutants having incorporated a non-naturallyencoded amino acid into the peptide sequence.

Purification of Biosynthetically Produced T20 and TEX Peptide Analogues

The resulting fusion peptides were biosynthetically produced inbacteria. Orthogonal tRNA and its specific orthogonal aminoacyl tRNAsynthetase were expressed to perform suppression of the T-20 or TEXconstructs. To avoid protein degradation in the bacterial cytoplasm,expression occurred by directing the fusion peptide into inclusionbodies (IB). The IBs containing the fusion peptides were resuspended inInclusion Body Resuspension Buffer (IBRB; 50 mM Tris, pH 7.5, 200 mMNaCl, 2 mM EDTA) containing 100 ug/ml lysozyme and 10 ug/ml DNase. Aftersix rounds of sonication of the resuspension, the samples werecentrifuged to spin down the pellets. The IB pellets were washed fourtimes to eliminate residual contaminants by sonication with InclusionBody wash buffer (50 mM Tris, pH 7.5, 30 mM NaCl, 1 mM EDTA) with 1%Triton X-100 and centrifugation between washes. Then the IB pellets werewashed twice by sonication with Inclusion Body wash buffer (50 mM Tris,pH 7.5, 100 mM NaCl, 1 mM EDTA) and centrifugation between washes. Thepellets were solubilized in Guanidinium Binding Buffer, pH 7.8 (6MGuanidine HCl, 20 mM NaPO4, pH 7.8, 500 nM NaCl) and bound toequilibrated ProBond resin for His-tag purification of the fusionpeptides. The resin was washed twice with Guanidinium Binding Buffer, pH7.8; twice with Guanidinium Wash Buffer, pH 6.0 (6M Guanidine HCl, 20 mMNaPO4, pH 6.0, 500 nM NaCl); and twice with Guanidinium Wash Buffer, pH5.3 (6M Guanidine HCl, 20 mM NaPO4, pH 5.3, 500 nM NaCl). The His-tagbound fusion peptides were eluted with Guanidinium Elution Buffer, pH4.0 (6M Guanidine HCl, 200 mM Acetic Acid, 20 mM NaPO4, pH 4, 500 nMNaCl).

Prior to sample lyophilization, a buffer exchange with GuanidiniumElution Buffer with 10% formic acid was performed using a PD-10desalting column. After lyophilization, the samples were thenresuspended in 70% formic acid for overnight cyanogen bromide (CNBr)cleavage. Since CNBr specifically cleaves C-terminal to methionine,cleavage with CNBr allows T-20 or TEX to be separated from its fusionpartner and further purified to obtain pure peptides for testing inanti-viral activity assays. Lane 4 of FIG. 9, Panel B shows the cleavageproducts of CNBr treatment. T-20 and the fusion partner (FP) areindicated with arrows. The other lanes were loaded as follows: lane1—marker, lane 2—before induction, lane 3—after induction.

After cleavage with CNBr, the samples were lyophilized and resuspendedin 8M urea and separated through preparative HPLC. The samples were runon a C8 prep HPLC column to purify away residual CNBr. The product waslyophilized and then resuspended in Guanidinium Binding Buffer, pH 7.8.The solubilized product was bound to equilibrated ProBond Resin, and theflow through was collected. The samples were then run on a C8 prep HPLCcolumn to purify the T-20 or TEX, and lyophilized. The purified peptideswere then resuspended in the following buffer: 22.5 mg/ml mannitol, 2.39mg/ml sodium carbonate, pH 9.

Mutations to Modify T20 and TEX

A selector codon was introduced into polynucleotides encoding both T-20and TEX analogue peptides to incorporate a non-naturally encoded aminoacid at designated conserved positions. The location of each selectorcodon was chosen based on the published crystal structure of the 6-helixbundle formation during HIV fusion. The selector codons were introducedby QuickChange mutagenesis according to manufacturer's instructions(Stratagene) and were confirmed by the sequencing of each individualmutant.

Five different constructs of T-20 were generated with a selector codonencoding a substitution with a non-naturally encoded amino acid. FIG. 10shows a map of the five residues of T-20 encoded by codons that weresubstituted with an amber codon: Threonine designated as T20 639; SerineT20 640; Leucine T20 641; Leucine T20 645; and Asparagine T20 651.

Eleven different constructs of TEX were generated with a selector codonencoding a substitution with a non-naturally encoded amino acid. FIG. 10also shows a map of the eleven residues of TEX encoded by codons thatwere substituted with an amber codon: Tryptophan designated as TEX 631;Aspartic acid designated as TEX 632; Isoleucine designated as TEX 635;Asparagine designated as TEX 636; Asparagine designated as TEX 637,Tyrosine designated as TEX 638; Threonine designated as TEX 639; Serinedesignated as TEX 640; Leucine designated as TEX 641; Leucine designatedas TEX 645; and Asparagine designated as TEX 651. FIG. 12 showssuppression occurred in both T20 651 (Panel A) and TEX 636 (Panel B).sup. is the abbreviation for suppressed. FIG. 12, Panels C and D showWestern blots of the samples run in FIG. 12, Panels A and B. Panel Eshows the residues substituted with p-acetyl-phenylalanine withasterisks in T-20 (T-20-Mut651) and in TEX (TEX-Mut636).

FIG. 5 shows TEX-W631, TEX-D632, TEX-N636, and TEX-T639 substituted withpara-acetyl phenylalanine.

Example 26

This example describes methods to measure biological activity of T-20comprising a non-naturally encoded amino acid.

In Vitro Fusion Assay to Test T20 and TEX Antiviral Activity

To evaluate T20 or TEX antiviral activity, a fusion assay is used basedon single-cycle infectivity. A schematic representation of the assay isshown in FIG. 11. Briefly, 293-T cells are cotransfected with twoplasmids: one plasmid that expresses only an HIV envelope gene (JRFL orJC2 env), and a second plasmid expressing a modified HIV proviral DNAthat carries the luciferase gene in place of HIV Nef gene and does notexpress its endogenous envelope gene (pHIV.Luc). Such pseudotyped envHIV-Luc virus is able to infect target cells only by one round ofinfection. HIV is produced 48 hours postransfection and is collected inthe supernatant of transfected cells. Quantitation of the virus is madeby measuring p24^(gag) by ELISA. Once the HIV concentration isdetermined, human target cells expressing human CD4 receptor and eitherone of the two human coreceptors CCR5 or CXCR4 are infected at differentMOI in the presence or absence of T20, TEX and their correspondingmutants. The cells are lysed three days post infection, and loaded withsubstrate to determine luciferase activity measured by an illuminometer.This assay is quantitative and the inhibition level of HIV fusion ofdifferent peptides is evaluated. FIG. 6 shows a schematic of the T20 orTEX activity assay. The results of this assay performed using T-20, TEX,and TEX mutants TEX-W631, TEX-D632, TEX-N636, and TEX-T639 substitutedwith para-acetyl phenylalanine, compared with FUZEON, are shown in FIG.7A and FIG. 7B. FIG. 8 shows the PEGylation of TEX-N636 with 5K and 30KPEG, conjugated as described in Example 3.

Alternatively, a number of other assays including but not limited to,other assays measuring antiviral activity, including but not limited to,assays measuring viral entry or viral fusion, known to one skilled inthe art may be used to monitor the activity of T-20 or TEX polypeptidesof the invention. Modifications to these assays to test combinationtherapy with another antiviral agent are also known to one skilled inthe art.

Also, standard methods which are well-known to those of skill in the artmay be utilized for assaying non-retroviral activity. See, for example,Pringle et al. (Pringle, C. R. et al., 1985, J. Medical Virology17:377-386) for a discussion of respiratory syncytial virus andparainfluenza virus activity assay techniques. Further, see, forexample, “Zinsser Microbiology”, 1988, Joklik, W. K. et al., eds.,Appleton & Lange, Norwalk, Conn., 19th ed., for a general review of suchtechniques. These references are incorporated by reference herein in itsentirety.

Animal studies may be performed with T-20 polypeptides of the invention.Such studies include, but are not limited to, toxicity studies.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference herein intheir entirety for all purposes.

TABLE 3 SEQ ID # Notes 1 amino acid sequence of RSV HR-Cgepiinyydplvfpsdefdasisqvnekinqslafirrsdellhnvntgkstt 2CCGGCGGTAGTTCAGCAGGGCAGAACGGCGGACTCTAAATCCGCATGGC M. jannaschii tRNAGCTGGTTCAAATCCGGCCCGCCGGACCA mtRNA_(CUA) ^(Tyr)  3CCCAGGGTAGCCAAGCTCGGCCAACGGCGACGGACTCTAAATCCGTTCT HLAD03; an optimizedtRNA CGTAGGAGTTCGAGGGTTCGAATCCCTTCCC TGGGACCA amber supressor tRNA 4GCGAGGGTAGCCAAGCTCGGCCAACGGCGACGGACTTCCTAATCCGTTC HL325A; an optimizedtRNA TCGTAGGAGTTCGAGGGTTCGAATCCCTCCCCTCGCACCA AGGA frameshiftsupressor tRNA 5 MDEFEMIKRNTSEIISEEELREVLKKDEKSAGIGFEPSGKIHLGHYLQIKKMIDAminoacyl tRNA RS LQNAGFDIIILLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSsynthetase for the TFQLDKDYTLNVYRLALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNTincorporation of p-azido-YYYLGVDVAVGGMEQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSS L-phenylalanineKGNFIAVDDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKRPEKF p-Az-PheRS(6)GGDLTVNSYEELESLFKNKELHPMDLKNAVAEELIKILEPIRKRL 6MDEFEMIKRNTSEIISEEELREVLKKDEKSAGIGFEPSGKIHLGHYLQIKKMID Aminoacyl tRNA RSLQNAGFDIIILLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGS synthetase for theSFQLDKDYTLNVYRLALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNT incorporation of p-SHYLGVDVAVGGMEQRKIHMLARELLPICKVVCIHNPVLTGLDGEGKMSSSbenzoyl-L-phenylalanineKGNFIAVDDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKRPEKF p-BpaRS(1)GGDLTVNSYEELESLFKNKELHPMDLKNAVAEELIKILEPIRKRL 7MDEFEMIKRNTSEIISEEELREVLKKDEKAAIGFEPSGKIHLGHYLQIKKMIDL Aminoacyl tRNA RSQNAGFDIIILLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSP synthetase for theFQLDKDYTLNVYRLALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNAI incorporation ofYLAVDVAVGGMEQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGpropargyl-phenylalanineNFIAVDDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKRPEKFGG Propargyl-PheRSDLTVNSYEELESLFKNKELHPMDLKNAVAEELIKILEPIRKR L 8MDEFE MIKRN TSEII SEEEL REVLK KDEKS AAIGF EPSGK IHLGH YLQIKAminoacyl tRNA RS KMIDL QNAGF DIIIL LADLH AYLNQ KGELD EIRKI GDYNK KVFEAsynthetase for the MGLKA KYVYG SPFQL DKDYT LNVYR LALKT TLKRA RRSME LIAREincorporation of DENPK VAEVI YPIMQ VNIPY LPVD VAVGG MEQRK IHMLA RELLPpropargyl-phenylalanineKKVVC IHNPV LTGLD GEGKM SSSKG NFIAV DDSPE EIRAK IKKAY Propargyl-PheRSCPAGV VEGNP IMEIA KYFLE YPLTI KRPEK FGGDL TVNSY EELESLFKNK ELHPM DLKNA VAEEL IKILE PIRKR L 9MDEFE MIKRN TSEII SEEEL REVLK KDEKS AAIGF EPSGK IHLGH YLQIKAminoacyl tRNA RS KMIDL QNAGF DIIIL LADLH AYLNQ KGELD EIRKI GDYNK KVFEAsynthetase for the MGLKA KYVYG SKFQL DKDYT LNVYR LALKT TLKRA RRSME LIAREincorporation of DENPK VAEVI YPIMQ VNAIY LAVD VAVGG MEQRK IHMLA RELLPpropargyl-phenylalanineKKVVC IHNPV LTGLD GEGKM SSSKG NFIAV DDSPE EIRAK IKKAY Propargyl-PheRSCPAGV VEGNP IMEIA KYFLE YPLTI KRPEK FGGDL TVNSY EELESLFKNK ELHPM DLKNA VAEEL IKILE PIRKR L 10MDEFEMIKRNTSEIISEEELREVLKKDEKSATIGFEPSGKIHLGHYLQIKKMID Aminoacyl tRNA RSLQNAGFDIIILLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGS synthetase for theNFQLDKDYTLNVYRLALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNincorporation of p-azido-PLHYQGVDVAVGGMEQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSS phenylalanineSKGNFIAVDDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKRPEKF p-Az-PheRS(1)GGDLTVNSYEELESLEKNKELHPMDLKNAVAEELIKILEPIRKRL 11MDEFEMIKRNTSEIISEEELREVLKKDEKSATIGFEPSGKIHLGHYLQIKKMID Aminoacyl tRNA RSLQNAGFDIIILLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGS synthetase for theSFQLDKDYTLNVYRLALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNPincorporation of p-azido-LHYQGVDVAVGGMEQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSS phenylalanineKGNFIAVDDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKRPEKF p-Az-PheRS(3)GGDLTVNSYEELESLEKNKELHPMDLKNAVAEELIKILEPIRKRL 12MDEFEMIKRNTSEIISEEELREVLKKDEKSALIGFEPSGKIHLGHYLQIKKMID Aminoacyl tRNA RSLQNAGFDIIILLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGS synthetase for theTFQLDKDYTLNVYRLALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNPincorporation of p-azido-VHYQGVDVAVGGMEQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSS phenylalanineKGNFIAVDDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKRPEKF p-Az-PheRS(4)GGDLTVNSYEELESLFKNKELHPMDLKNAVAEELIKILEPIRKRL 13MDEFEMIKRNTSEIISEEELREVLKKDEKSATIGFEPSGKIHLGHYLQIKKMID Aminoacyl tRNA RSLQNAGFDIIILLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGS synthetase for theSFQLDKDYTLNVYRLALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNPincorporation of p-azido-SHYQGVDVAVGGMEQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSS phenylalanineKGNFIAVDDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKRPEKF p-Az-PheRS(2)GGDLTVNSYEELESLFKNKELHPMDLKNAVAEELIKILEPIRKRL 14MDEFEMIKRNTSEIISEEELREVLKKDEKSALIGFEPSGKIHLGHYLQIKKMID Aminoacyl tRNA RSLQNAGFDIIILLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGS synthetase for theEFQLDKDYTLNVYRLALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVN incorporation of p-GCHYRGVDVAVGGMEQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSS acetyl-phenylalanineSKGNFIAVDDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKRPEKF (LW1)GGDLTVNSYEELESLFKNKELHPMDLKNAVAEELIKILEPIRKRL 15MDEFEMIKRNTSEIISEEELREVLKKDEKSALIGFEPSGKIHLGHYLQIKKMID Aminoacyl tRNA RSLQNAGFDIIILLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGS synthetase for theEFQLDKDYTLNVYRLALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVN incorporation of p-GTHYRGVDVAVGGMEQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSS acetyl phenylalanineSKGNFIAVDDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKRPEKF (LW5)GGDLTVNSYEELESLFKNKELHPMDLKNAVAEELIKILEPIRKRL 16MDEFEMIKRNTSEIISEEELREVLKKDEKSAAIGFEPSGKIHLGHYLQIKKMID Aminoacyl tRNA RSLQNAGFDIIILLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGS synthetase for theEFQLDKDYTLNVYRLALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVN incorporation of p-GGHYLGVDVIVGGMEQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSS acetyl-phenylalanineKGNFIAVDDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKRPEKF (LW6)GGDLTVNSYEELESLFKNKELHPMDLKNAVAEELIKILEPIRKRL 17MDEFEMIKRNTSEIISEEELREVLKKDEKSAAIGFEPSGKIHLGHYLQIKKMID Aminoacyl tRNA RSLQNAGFDIIILLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGS synthetase for theRFQLDKDYTLNVYRLALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNincorporation of p-azido-VIHYDGVDVAVGGMEQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSS phenylalanineSKGNFIAVDDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKRPEKF (AzPheRS-5)GGDLTVNSYEELESLFKNKELHPMDLKNAVAEELIKILEPIRKRL 18MDEFEMIKRNTSEIISEEELREVLKKDEKSAGIGFEPSGKIHLGHYLQIKKMID Aminoacyl tRNA RSLQNAGFDIIILLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGS synthetase for theTFQLDKDYTLNVYRLALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNTincorporation of p-azido-YYYLGVDVAVGGMEQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSS phenylalanineKGNFIAVDDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKRPEKF (AzPheRS-6)GGDLTVNSYEELESLFKNKELHPMDLKNAVAEELIKILEPIRKRL 19GAGTGGGATAGAGAAATTAACAATTACACAAGTTTAATACACTCCTTAA Nucleic AcidTTGAAGAATCGCAAAACCAGCAAGAAAAGAATGAACAAGAATTATTGG Encoding TEXAATTAGATAAATGGGCAAGTTTG TGGAATTGGTTT 20E W D R E I N N Y T S L I H S L I E E S Q N Q Q E K N E Q E TEX peptideL L E L D K W A S L W N W F 21ATGAGCGATAAAATTATTCACCTGACTGACGACAGTTTTGACACGGATGT Nucleic AcidACTCAAAGCGGACGGGGCGATCCTCGTCGATTTCTGGGCAGAGTGGTGC Encoding Trx-GGTCCGTGCAAAATGATCGCCCCGATTCTGGATGAAATCGCTGACGAAT TEV-TEXATCAGGGCAAACTGACCGTTGCAAAACTGAACATCGATCAAAACCCTGG fusionCACTGCGCCGAAATATGGCATCCGTGGTATCCCGACTCTGCTGCTGTTCAAAACGGTGAAGTGGCGGCAACCAAAGTGGGTGCACTGTCTAAAGGTCAGTTGAAAGAGTTCCTCGACGCTAACCTGGCCGGTTCTGGTTCTGGCCATATGCACCATCATCATCATCATTCTTCTGGTGAAAACCTGTACTTCCAA(AGC)GAGTGGGATAGAGAAATTAACAATTACACAAGTTTAATACACTCCTTAATTGAAGAATCGCAAAACCAGCAAGAAAAGAATGAACAAGAATTATTGGAATTAGATAAATGGGCAAGTTTG TGGAATTGGTTT 22M S D K I I H L T D D S F D T D V L K A D G A I L V D F W A Trx-TEV-TEXE W C G P C K M I A P I L D E I A D E Y Q G K L T V A K L NFusion PeptideI D Q N P G T A P K Y G I R G I P T L L L F K N G E V A A TK V G A L S K G Q L K E F L D A N L A G S G S G H M H H HH H H S S G E N L Y F Q S - TEV site E W D R E I N N Y T S L IH S L I E E S Q N Q Q E K N E Q E L L E L D K W A S L W N W F 23ATGGAATGGGATCGTGAAATCAACAACTACACAAGCTTAATACACAGCT Nucleic AcidTAATTGAGGAGAGCCAGAACCAGCAGGAGAAAAATGAGCAGGAACTGT Encoding 2TEXTGGAACTGGATAAATGGGCAAGCCTGTGGAATTGGTTTGGTGGTGGCTCT PeptideGGCGGTGGTAGCGGTGGCGGTAGTGAGTGGGATAGAGAAATTAACAATTACACAAGTTTAATACACTCCTTAATTGAAGAATCGCAAAACCAGCAAGAAAAGAATGAACAAGAATTATTGGAATTAGATAAATGGGCAAGTTTG TGGAATTGGTTT 24M E W D R E I N N Y T S L I H S L I E E S Q N Q Q E K N E Q 2TEX PeptideE L L E L D K W A S L W N W F G G S G G G S G G G S -linker EW D R E I N N Y T S L I H S L I E E S Q N Q Q E K N E Q E LL E L D K W A S L W N W F

1. A biosynthetic polypeptide fusion inhibitors (BPFI) comprising one or more non-naturally encoded amino acids, wherein the BPFI is included in a construct comprising a fusion partner linked to a methionine which is linked to said BPFI.
 2. The BPFI of claim 1, wherein the BPFI comprises one or more post-translational modifications.
 3. The BPFI of claim 1, wherein the polypeptide is linked to a linker, polymer, or biologically active molecule.
 4. The BPFI of claim 3, wherein the polypeptide is linked to a water soluble polymer.
 5. The BPFI of claim 1, wherein the polypeptide is linked to a bifunctional polymer, bifunctional linker, or at least one additional BPFI.
 6. The BPFI of claim 5, wherein the bifunctional linker or polymer is linked to a second polypeptide.
 7. The BPFI of claim 6, wherein the second polypeptide is a BPFI.
 8. The BPFI of claim 4, wherein the water soluble polymer comprises a poly(ethylene glycol) moiety.
 9. The BPFI of claim 4, wherein said water soluble polymer is linked to a non-naturally encoded amino acid present in said BPFI.
 10. The BPFI of claim 1, wherein the non-naturally encoded amino acid is reactive toward a linker, polymer, or biologically active molecule that is otherwise unreactive toward any of the 20 common amino acids in the polypeptide. 11-40. (canceled)
 41. The BPFI of claim 1, wherein the BPFI is selected from the group comprising SEQ ID NO: 20, SEQ ID NO: 22, and SEQ ID NO:
 24. 